Angiogenesis reagents: Novus Biologicals

NB100-105

HIF-1 alpha Antibody (H1alpha67)

(45 Reviews)

(1043 Publications)

0.1 ml

NB100-105

HIF-1 alpha induction by CoCl2 on Caki-1 cell lysate. WB image submitted by a verified customer review.

<b>Biological Strategies Validation and Genetic Strategies Validation.</b>HIF-1 alpha was detected in immersion fixed DFO treated Hela cells (left) but was not detected in HIF-1 knockout HeLa cells (right) using Mouse Anti-human HIF-1 alpha monoclonal antibody (Catalog # NB100-105) at 25 ug/mL for 3 hours at room temperature. Cells were stained using a NorthernLights (TM) 557-conjugated Donkey Anti-Mouse IgG Secondary Antibody (red; Catalog # NL007) and counterstained with DAPI (blue). Specific staining was localized to nuclei.

<b>Biological Strategies Validation and Genetic Strategies Validation.</b>Naive CD4 T cells from WT, VHL-deficient (Vhl KO), or HIF-1 alpha-deficient (HIF-1 alpha KO) mice were differentiated under IL-22-skewing conditions for a total of 60 h. Some cells remained at normoxia for the duration of the culture (N); others were at normoxia for 35 h and then hypoxia (1% O2) for 24 h (H). At 60 h, nuclear extracts were harvested, and HIF-1 alpha and Lamin B1 levels were analyzed by Western blot. Image from verified customer review.

Analysis using the Alexa Fluor (R) 488 conjugate of NB100-105. Staining of HIF-1 alpha in multiple myeloma cells: H929 cells (0.5 x 10^6) were stained with Alexa Fluor 488(R) conjugated HIF-1 alpha antibody (NB100-105AF488). Image courtesy of Dr. Barbara Muz at Washington University in St. Louis School of Medicine.

Staining of HIF-1 alpha in human kidney using NB100-105. Renal tubular epithelium showed moderate membranous, cytoplasmic and nuclear staining, and glomeruli showed faint to moderate nuclear staining.

HIF1alpha/PFKFB3 is upregulated in beta-cells of HIP rats and humans with type 2 diabetes. Representative Western blot of PFKFB3 and HIF1alpha levels in nuclear-enriched- and whole cell extracts from non-diabetic (ND) and T2D donor islets. Data are presented as mean +/- SEM, n = 3 independent biological samples for each group. Statistical significance was analyzed by Student t-test (*p 50 myofibers from 3 mice/group/time point) & stained for Pax7, DAPI, & EdU (A), HIF2A (B), or HIF1A (C). Scale bars: 20 μm. Arrowheads indicate SCs. (D) Number of Pax7+ SCs per myofiber at various time points. (E) Percentage of EdU+ SCs at various time points. (F) Percentage of HIF2A+ SCs at various time points. (G) Percentage of HIF1A+ SCs at various time points. Data represent the mean ± SEM. Image collected & cropped by CiteAb from the following publication (//www.jci.org/articles/view/96208), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: HIF-1 alpha Antibody (H1alpha67) [NB100-105] - mTORC2 not mTORC1 regulates HIF‐2 alpha , GOT1 in PDAC under prolonged hypoxia. (A & B) HIF‐2 alpha mRNA & protein levels determined by qRT‐PCR (mRNA) & Western blot (protein) after treatment of Panc‐1 & Capan‐2 cells with mTORC1 inhibitor rapamycin for 48 hrs at 3% or 1% O2. beta ‐Actin was used as loading control. (C & D) Panc‐1 & Capan‐2 cells were treated with mTORC1/mTORC2 inhibitor PP242 & cultured for 48 hrs at 20%, 3% or 1% O2, & HIF‐2 alpha , GOT1 mRNA & protein levels were determined by qRT‐PCR (mRNA) & Western blot (protein). beta ‐Actin was used as loading control. Data are presented as mean ± S.D. from three independent experiments. *P 50 myofibers from n = 3 mice) showing that nmGFP+ QSCs were pimonidazole+. Scale bars: 50 μm & 10 μm (insets). Inset images show that pimonidazole signals were relatively enriched in the cytoplasm of QSCs. Arrowheads indicate a QSC; asterisks indicate a myonucleus. (B) Percentage of pimonidazole+ QSCs. (C) Timeline of in vivo CCI-103F labeling in C57BL/6 mice & representative images of uninjured/resting EDL myofibers (n >50 myofibers from 3 mice) showing that nmGFP+ QSCs were CCI-103F+. Arrowheads indicate a QSC; asterisks indicate a myonucleus. Scale bar: 20 μm. (D) Percentage of CCI-103F+ QSCs. (E & F) Representative images of uninjured/resting EDL myofibers from C57BL/6 mice (n >50 myofibers from 6 mice/group) showing that most QSCs were HIF2A+, but HIF1A–. Scale bars: 10 μm. (G) Percentage of HIF1A+ & HIF2A+ QSCs. Data represent the mean ± SEM. Image collected & cropped by CiteAb from the following publication (//www.jci.org/articles/view/96208), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: HIF-1 alpha Antibody (H1alpha67) [NB100-105] - FDFT1 inhibits glycolysis through suppressing AKT-mTOR-HIF1 alpha pathway in CRC.aFDFT1 overexpression reduced glucose uptake in CT26 cells. bFDFT1 overexpression decreased lactate production via glycolysis in CT26 cells. c ECAR reduced when FDFT1 overexpressed in CT26 cells. d OCR increased when FDFT1 overexpressed in CT26 cells. eFDFT1 knockdown increased glucose uptake in CT26 cells. fFDFT1 knockdown increased lactate production via glycolysis in CT26 cells. g ECAR increased when FDFT1 knocked down in CT26 cells. h OCR decreased when FDFT1 knocked down in CT26 cells. i, mFDFT1 overexpression inhibited protein & mRNA expression of mTOR-targeted glycolytic enzymes, including GLUT1, HK2, PGK1, GPI, & LDHA, in CT26 cells. j, mFDFT1 knockdown increased protein & mRNA expression of mTOR-targeted glycolytic enzymes, including GLUT1, HK2, PGK1, GPI, & LDHA, in CT26 cells. k, nFDFT1 overexpression decreased protein & mRNA expression of AKT, mTOR, & HIF1 alpha . l, nFDFT1 knockdown increased protein & mRNA expression of AKT, mTOR, & HIF1 alpha . o Photograph of dissected tumors (the first line: normal diet, second line: FMD + glucose, third line: FMD, n = 5). p The tumor volumes measured every 2 days after 13th day. The FMD + glucose group can reverse tumor growth inhibition induced by FMD (n = 5; ns: P = 0.1838; P = 0.0001). q The protein level of FDFT1 & mTOR in dissected tumor samples from normal diet group, FMD group & FMD + glucose group measured by western blotting. r The glucose level in these three groups. Error bars, mean ± SD, data are from three independent experiments. Two-sided t tests. *P < 0.05, **P < 0.01, ***P < 0.001, compared w/ control group (or normal diet group). #P < 0.05, ##P < 0.01. Image collected & cropped by CiteAb from following publication (//pubmed.ncbi.nlm.nih.gov/32313017), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: HIF-1 alpha Antibody (H1alpha67) [NB100-105] - Hypoxia induces expression of WNT11 through VHL.(A,B) Higher basal levels of WNT11 protein in Vhl-deleted cells (lenti-Cre infected Vhlf/f). EMSCs isolated from Vhlf/f mouse were infected with lentivirus carrying either GFP gene (for control) or Cre recombinase (for knockout). Non-infected cells were also used as a control. Immunoblot analysis of control or Vhl KO EMSCs treated with 0.1 mM DMOG (A), & EMSCs exposed to air (21% O2) or hypoxia (1% O2) for 24 hrs (B). Laminin, alpha -tubulin, & lamin A/C were used as loading controls, WNT11 normalized to alpha -Tubulin was shown. (C,D) Inactivation of the Vhl gene results in increased Wnt11 mRNA. Wnt11 & Vegf mRNA levels in liver (C) or duodenum (D) were measured by qPCR in Liver-VhlcKO or duodenum-VhlcKO & control mice (n = 5 per group). Values normalized to Tbp mRNA are expressed relative to tissues from control mice. For panels (C,D), values are mean ± s.e.m. *p < 0.05, **p < 0.01. Image collected & cropped by CiteAb from the following publication (//www.nature.com/articles/srep21520), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

$Western Blot: HIF-1 alpha Antibody (H1alpha67) [NB100-105] - Role of HIF-1 alpha , FOXO3 in hypoxia-induced BNIP3 expression. (A) UCB-hMSCs were incubated with various durations of hypoxia (0–48 h). The protein expressions of HIF-1 alpha & beta -actin were detected by western blot. n = 4. (B) UCB-hMSCs were pretreated with NAC (5 mM) for 30 min prior to hypoxia incubation for 24 h. The protein expressions of HIF-1 alpha , lamin A/C & beta -tubulin in non-nuclear & nuclear fractionized cell samples were assessed by using western blot. n = 3. (C) UCB-hMSCs were immuno-stained with HIF-1 alpha & PI (magnification ×600). Scale bars, 37.5 µm. (D) HIF1A siRNA or NT siRNA was transfected to cells prior to hypoxia treatment for 24 h. The mRNA expression of BNIP3 was analyzed by qPCR. n = 6. (E) The protein expressions of BNIP3 & HIF-1 alpha were detected by western blot. n = 4. (F) NAC (5 mM) was pretreated to UCB-hMSCs prior to hypoxia treatment for 24 h. FOXO3, lamin A/C & beta -tubulin proteins expressions were assessed by western blot. n = 3. (G) FOXO3 siRNA transfected to UCB-hMSCs prior to hypoxia treatment for 24 h. The FOXO3 mRNA expression was measured by qPCR. n = 6. (H) BNIP3, FOXO3 & beta -actin expressions were detected by western blot. n = 3. Western blot data were normalized by beta -actin, & qPCR data were normalized by ACTB mRNA expression level. Lamin A/C & beta -tubulin were used as nuclear & non-nuclear protein controls, respectively. Quantitative data are presented as a mean ± S.E.M. All blots & confocal images are representative. *p < 0.05 versus control, #p < 0.05 versus hypoxia. Image collected & cropped by CiteAb from the following publication (//linkinghub.elsevier.com/retrieve/pii/S2213231717303804), licensed under a CC-BY license. Not internally tested by Novus Biologicals.$

Western Blot: HIF-1 alpha Antibody (H1alpha67) [NB100-105] - ROS overproduction mediated apoptosis induced by combined treatment of DDP & HIF-1 alpha in PC-3 xenografts & cell culture. (a) ROS monitored by DHE (red) & nuclei by DAPI (blue) staining in PCa xenografts (scale bars, 50 µm). (b) MDA formation of PCa xenografts was examined after various treatments (c,d). PC-3 cells were treated with DDP, si-HIF-1 alpha plasmid, or both, in the presence or absence of NAC (5 mM) or DHLA (0.25 mM) for 24 h. Total lysates (c) & culture media (d) were used to detect cellular H2O2 level. (e,f) Western analysis for HIF-1 alpha as well as cleaved caspase-3 & PARP in PC-3 cells following various treatments. Data were presented as mean ± SD of three independent experiments. *p < 0.05 versus control group; #p < 0.05 versus si-HIF-1 alpha or DDP group; $p < 0.05 versus DDP/si-HIF-1 alpha group. The original blots are presented in Supplementary Figure 8. Image collected & cropped by CiteAb from the following publication (//www.nature.com/articles/s41598-017-07973-4), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: HIF-1 alpha Antibody (H1alpha67) [NB100-105] - miR-128 downmodulation regulates MAFG increase in hypoxic condition both in vitro & in vivo. C2C12 cells exposed to hypoxia (2% O2) for 4 h, & miR-128 levels (a) determined by RT-qPCR on total RNAs after normalization w/ the small RNU6. Data are expressed as relative to values obtained in untreated control which set equal to 1. Each column in the panels represents the mean ± SD of 3 independent experiments. ∗p < 0.05. Western blotting analysis (b) of HIF-1 alpha , MAFG, & HMOX-1 performed on total extracts from the same cells, as described in Materials & Methods. Tubulin used as a loading control. mRNA levels of HMOX-1 & x-CT genes (c) determined by RT-qPCR on total RNAs, & relative changes calculated by comparing treated cells versus control, after normalization w/ c-ABL. Data reported as relative to values obtained in normoxia, which set equal to 1. Each column in the panel represents the mean ± SD of 3 independent experiments. ∗p < 0.05. Representative laser Doppler analysis (d) of blood flow before & 4 hours after (post 4 h) hindlimb ischemia procedure (n = 3-4 mice/group). Representative Western blot analysis & densitometric analysis (e) of MAFG & HMOX-1 protein levels performed on hindlimb muscle lysates from littermates after sham procedure (sham) or not-ischemic limb (NIL) & ischemic limb (IL) 4 h after femoral artieriectomy. Tubulin protein levels used as loading controls (∗p < 0.05 versus sham; n = 3-4 hindlimb/group). miR-128 levels (f) determined by RT-qPCR on total RNAs after normalization w/ the small RNU6. The values obtained in not-ischemic limb (NIL) set equal to 1. The data are expressed as the mean ± standard error & are representative of 3 independent experiments. ∗p < 0.05. Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/29138682), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: HIF-1 alpha Antibody (H1alpha67) [NB100-105] - Effect of ENMD-1198 on HIF-1 alpha & VEGF expression in HCC cells. To determine effects of ENMD-1198 on nuclear HIF-1 alpha expression, HUH-7 cells were incubated for 16 h ± ENMD-1198 & subsequently exposed to hypoxia (1% O2, 4 h). A) Western blot analysis of whole protein showed that ENMD-1198 effectively blunted hypoxic induction of HIF-1 alpha protein. B) In addition, treatment with ENMD-1198 down-regulated constitutive VEGF-A mRNA levels in HCC cells (HepG2), as measured by real-time PCR (* P < 0.01) (n = 3/group). VEGF-A mRNA expression is normalized to beta -actin. C) ELISA analysis for VEGF in culture supernatants (HepG2). Hypoxia (20 h, 1% O2) markedly increased VEGF protein. ENMD-1198 treatment lowered VEGF secretion under hypoxic conditions (*P < 0.05). D) Western blot analysis for VEGF. Cells were incubated under either non-hypoxic, or hypoxic conditions ± ENMD-1198. Treatment with ENMD-1198 slightly lowered the hypoxic induction of VEGF, as determined by densitometry. Bars: SEM. Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/18651980), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: HIF-1 alpha Antibody (H1alpha67) [NB100-105] - Effects of resveratrol on the expression of SIRT1, PGC-1 alpha , HIF-1 alpha & apoptosis proteins in vitro. (A) The expression of SIRT1, PGC-1 alpha , & Bcl-2 proteins were lower in iohexol treated HK-2 cells compared with control cells, which was reversed by treatment with resveratrol. The expression of HIF-1 alpha , Bax, cleaved caspase-3, & Cyt-C were markedly increased after iohexol injection, which was reversed by treatment with resveratrol. Furthermore, this effect was enhanced by HIF-1 alpha inhibitor, 2-MeOE2. (B–H) Relative densitometry of SIRT1–PGC-1 alpha –HIF-1 alpha signaling proteins expressed as mean ± standard error. *P < 0.05 vs. Cont; #P < 0.05 vs. PC–AKI. Cont, control; Res, resveratrol; PC–AKI, post-contrast acute kidney injury; SIRT1, silent information regulator l; HIF-1 alpha , hypoxia-inducible transcription factor-1 alpha ; Cyt-C, cytochrome C; PGC-1 alpha , peroxisome proliferator-activated receptor gamma coactivator-1 alpha; Ex527, SIRT1 specific inhibitor; LG, low glucose. Groups: Res, HG+res+iohexol; Ex527, HG+Ex527+iohexol; PC–AKI, HG+iohexol; 2-MeOE2, HG+res+2-MeOE2+iohexol; Cont, LG. Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/31402864), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: HIF-1 alpha Antibody (H1alpha67) [NB100-105] - Upregulation of HIF-1 alpha in human PCa. (a) HIF-1 alpha protein was detected by western blot in nonmalignant (RWPE-1 & BPH1) & PCa cell lines (PC-3, DU145, LNCaP, & 22RV1) as indicated. (b–d) Total RNA extracted from RWPE-1, BPH1, PC-3, DU145, LNCaP, & 22RV1 cells was subjected to qRT-PCR for HIF-1 alpha (b), VEGF (c) & GLUT4 (d). (e) The HIF-1 alpha promoter-driven reporter (firefly luciferase) & a control vector (Renilla luciferase) were co-transfected into RWPE-1, BPH1, PC-3, DU145, LNCaP, & 22RV1 cells for measurement of luciferase activity. HIF-1 alpha promoter activity was calculated as a ratio of firefly to Renilla activity. (f) Human normal & malignant tissue (Gleason score 9) sections were probed with HIF-1 alpha antibody (scale bars, 100 µm). (g) Protein expression of HIF-1 alpha , VEGF, & GLUT4 were examined with western blot, in PC-3, DU145, & LNCaP cells after various treatments as indicated. Data are expressed as mean ± SD of seven independent experiments. $p < 0.05 versus RWPE-1 or BPH1 cells or normal tissue. *p < 0.05 versus control group. #p < 0.05 versus si-HIF-1 alpha or DDP group. Original blots are shown in Supplementary Figure 5. C: Ctrl; D: DDP; S: si-HIF-1 alpha ; D/S: DDP/si-HIF-1 alpha . Image collected & cropped by CiteAb from the following publication (//www.nature.com/articles/s41598-017-07973-4), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: HIF-1 alpha Antibody (H1alpha67) [NB100-105] - Cx43-overexpressing cells regulate the expression of HIF-1 alpha , vascular endothelial growth factor (VEGF), & the proliferation of endothelial cells. (a) The expression of Cx43 & HIF-1 alpha was measured in Cx43-overexpressing cells by Western blot analysis. beta -actin expression served as loading controls for & total protein. Inserted values indicated relative proteins expression in comparison with beta -actin; (b) Cx43-overexpressing cells were cotransfected with pCLNCX-6× HRELuc & pTCYLacZ plasmids. At 6 h post-transfection, their luciferase activities were determined & normalized with beta -gal activity. Data shown were the mean ± SD (n = 4); (c) The conditioned medium of Cx43-overexpressing cells was measured by ELISA. Data shown were the mean ± SD (n = 4); & (d) The conditioned medium of Cx43-overexpressing cells reduced the proliferation of endothelial cells. The HEMC-1 cells treated with conditioned medium of Cx43-overexpressing cells were examined the proliferation activity. Cell viability was assessed by the WST-1 assay. Data shown were the mean ± SD (n = 4). * p < 0.05; *** p < 0.001. Image collected & cropped by CiteAb from the following publication (//www.mdpi.com/1422-0067/16/1/439), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: HIF-1 alpha Antibody (H1alpha67) [NB100-105] - Prolonged hypoxia increases glutamine metabolism in PDAC cells, & HIF‐2 alpha can promote non‐canonical glutamine metabolism in chronic hypoxic conditions. (A) Time course of glutamine consumption at 1%, 3% or 20% O2, each time data point is an average of triplicate experiments. (B) Panc‐1 & Capan‐2 were incubated for 48 hrs at 1%, 3% or 20% O2, GLS1, GOT1 & GOT2 protein were measured by Western blot. beta ‐Actin was used as loading control. (C) Panc‐1 & Capan‐2 were incubated for 48 hrs at 1%, 3% or 20% O2, GLS1, GOT1 & GOT2 mRNA were measured by qRT‐PCR. Data are presented as mean ± S.D. from three independent experiments. (D & F) Si‐HIF‐2 alpha ‐transfected Panc‐1 & Capan‐2 cultured at 1% or 3%O2 for 48 hrs. The level of HIF‐2 alpha & glutamine metabolism enzymes mRNA & protein were determined by qRT‐PCR (mRNA) & Western blot (protein), & beta ‐actin was used as loading control. Data are presented as mean ± S.D. from three independent experiments. *P < 0.05, **P < 0.01. Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/28544376), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: HIF-1 alpha Antibody (H1alpha67) [NB100-105] - HIF1 alpha & PFKFB3 are protective against hIAPP mediated beta -cell death. a Representative immunoblotting (left) & quantification (right) of the whole cell extract (WCE) from asynchronous INS 832/13 cells treated with either HIF1 alpha inhibitor KC7F2 (10 μM) or PFKFB3 siRNA (75 nM) for 56 h versus control. Cells were transduced with hIAPP adenovirus (75 MOI) or LacZ adenovirus as control (75 MOI) for last 32 h of culture. DMSO or CTRL siRNA, respectively were added to LacZ or hIAPP to equal DMSO or PFKFB3 siRNA used in experimental groups. GAPDH was used as a loading control. Data are presented as mean ± SEM, n = 4, *p < 0.05, **p < 0.01. b Quantification of DNA content distribution after FACS analysis of INS 832/13 cells treated as described in (a). Data are the mean ± SEM, n = 4, *p < 0.05, **p < 0.01. c The frequency of TUNEL positive INS 832/13 cells treated as described in (a). Data are the mean ± SEM, n = 4 in (a) & (b), & n = 3 independent experiments in (c). Statistical significance was analyzed by one-way ANOVA test with Tukey’s post-test (*p < 0.05, **p < 0.01). See Supplementary Fig. 10 for additional supportive data Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/31213603), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Immunohistochemistry: HIF-1 alpha Antibody (H1alpha67) [NB100-105] - Immunohistochemical & Western blot analysis of HIF-1 alpha & VEGF expression in OVA-challenged mice.(A & B) Immunostaining for HIF-1 alpha & VEGF in nasal mucosa following the last challenge. Positive HIF-1 alpha signals are brown & are predominantly nuclear & VEGF is predominantly cytoplasmic (magnification 400×, scale bar = 20 µm). (C & D) Representative Western blot analysis showing HIF-1 alpha & VEGF (with beta -actin as a loading control) expression in the nasal mucosa 24 h after the last challenge. All densitometric analyses are presented as the relative ratio of each molecule to beta -actin & the ratio in negative control mice was set to 100. Values represent means±SEM (n = 6 mice). *Significantly different from negative control, p<0.05; #significantly different from positive control, p<0.05. Image collected & cropped by CiteAb from the following publication (//dx.plos.org/10.1371/journal.pone.0048618), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Immunohistochemistry: HIF-1 alpha Antibody (H1alpha67) [NB100-105] - Quantification of MAOB, HiF-1 alpha , & GFAP levels in gliomas(A) Relative levels of MAOB in normal brain, five anaplastic astrocytomas (AA), a geministic astrocytoma (Gem), an astroblastoma (Ablast), & 20 GBM tumors, with the GBM tumors arranged from lowest to highest signal. The same left-right arrangement of GBM tumors is also used in (B) & (C). (B) Relative levels of HiF-1 alpha . (C) Relative levels of GFAP. (D) Correlation between MAOB & HiF-1 alpha in gliomas & GBMs. (E) Correlation between MAOB & GFAP in gliomas & GBMs. (F) Correlation between HiF-1 alpha & GFAP. The coefficient of determination, R2, & p values are shown on all correlation plots. All points are mean ± SD, with n = 4 for tumor samples & n = 12 for the control brain samples. Image collected & cropped by CiteAb from the following publication (//www.oncotarget.com/lookup/doi/10.18632/oncotarget.6582), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Immunohistochemistry: HIF-1 alpha Antibody (H1alpha67) [NB100-105] - DC101, evofosfamide, & HIF-1 alpha shRNA treatment of HT1080 sarcomasA. Western blot of HT1080 cells stably transduced with HIF-1 alpha shRNA (sh.HIF-1 alpha ) or scrambled control shRNA (sh.Scr) & grown in 21% oxygen or 1% oxygen. beta -actin serves as loading control. B. Growth of HT1080 cells stably transduced with sh.HIF-1 alpha or sh.Scr in athymic nude mice. Groups were treated with control IgG 20 mg/kg 3 times per week, DC101 20 mg/kg 3 times per week, and/or evofosfamide (Evo) 50 mg/kg 5 times per week. C. Photos of H&E staining, cleaved caspase 3 IF, cleaved caspase 3 & CD31 IF, CD31 IHC, pimonidazole IHC, & HIF-1 alpha IHC. D. Graphs of microvessel density, pimondizale area, & nuclear HIF-1a expression. Bars represent standard deviation. **p < 0.05 compared to all other groups. Image collected & cropped by CiteAb from the following publication (//www.oncotarget.com/lookup/doi/10.18632/oncotarget.10212), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: HIF-1 alpha Antibody (H1alpha67) [NB100-105] - WNT11 is induced by hypoxia or hypoxic mimetics in different cell types.(A) Increased Wnt11 mRNA in EMSC adipocytes (Day 12) after hypoxia-mimetic treatments. EMSC adipocytes were treated with CoCl2 (0.1 mM), DFO (0.1 mM) or DMOG (0.1 mM) for 24 hrs. Values were normalized to Tbp mRNA & are expressed relative to control (n = 3). (B,C) Increased Wnt11 mRNA by hypoxia in EMSC preadipocytes & adipocytes (Day 0–12 after differentiation) (B), & C2C12 myoblast & myocyte (Day 0 & 8 after differentiation) (C). Wnt11 mRNA was assessed by quantitative PCR in cells exposed to air (21% O2) or hypoxia (1% O2) for 24 hrs. (n = 4). Values were normalized to Tbp mRNA & are expressed relative to 21% O2 samples (left panel). (D) Immunoblot analyses of HeLa cells under normal air or hypoxia for 24 hrs. (E,F) Induction of Wnt11 by increasing concentrations of DMOG in MDA-MB-231 cells (E) & 4T1 cells (F). (G) EMSCs treated with 0.1 mM DMOG for the indicated times. Wnt11 & Vegf mRNA expression was measured by qPCR & normalized to Tbp mRNA (n = 4). (H) WNT11 protein levels after DMOG treatment normalized to alpha -Tubulin (upper panel; n = 4). Representative immunoblots of EMSCs treated with 0.1 mM DMOG for the indicated times (Lower panel). (I) Protein expression in MDA-MB-231 cells treated with 0.1 mM DMOG. (J) Induction of Wnt11 promoter activity by hypoxia or hypoxia mimetics. pGL3-Wnt11 promoter plasmid was transfected into C2C12 cells. Cells were incubated with DMOG (left panel, n = 4) or under 21% O2 or 1% O2 (right panel, n = 8) for 24 hrs. For panels (A–C,G,H,J), values are mean ± s.e.m. *p < 0.05, **p < 0.01. For panels of immunoblotting, laminin, alpha -tubulin, & ERK were used as loading controls, WNT11 normalized to alpha -Tubulin was shown. Image collected & cropped by CiteAb from the following publication (//www.nature.com/articles/srep21520), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: HIF-1 alpha Antibody (H1alpha67) [NB100-105] - CAPE inhibits MCT-induced HIF-1 alpha expression & PDGF-BB production in rats. (A) Rats were treated with PBS or CAPE (5 or 10 mg/kg) from days 14–28 after MCT treatment 14 days (60 mg/kg). Preparation of lung tissues was analyzed by Western blot to determine the levels of HIF-1 alpha protein. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH)was used as a loading control. (B) Serum levels of PDGF-BB in rat was assayed by an ELISA kit. (C) Representative images of immunohistochemical staining for HIF-1 alpha in lung sections from the rats. Scale bars, 50 μm. (D) Immunofluorescence staining of alpha-smooth muscle actin ( alpha -SMA) & HIF-1 alpha in lung sections from rats. Scale bars, 50 μm. Data in (A,B) are expressed as mean ± SEM of three independent experiments. *** p < 0.01, as compared with the PBS group. ### p < 0.01, as compared with the rats exposed to MCT alone. Results are representative of three rats per experimental group. Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/30909527), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: HIF-1 alpha Antibody (H1alpha67) [NB100-105] - mTOR expression is inversely correlated with FDFT1 expression.aFDFT1 overexpression inhibited the protein level of mTOR, whereas FDFT1 knockdown increased the protein level of mTOR in CT26 & SW620 cells. bFDFT1 expression & mTOR expression were negatively correlated in CRC patient samples. Scale bar: 200 µm. c The mTOR silencing efficiency of the siRNA in CT26 & SW620 cells was validated by western blotting. d The effect of mTOR silencing on FDFT1 expression level in CT26 was evaluated by western blotting. e The protein level of mTOR when FDFT1 knockdown combined with or without mTOR inhibitor in CT26. f The protein level of pS6k, S6k, pS6, & S6 under the effect of fasting & FDFT1 overexpression in CRC cells. g, h CCK8 proliferation assays showed that the silencing of mTOR decreased the proliferation of CT26 & SW620 cells. k, n The silencing of mTOR reduced glucose uptake & lactate production in CT26 & SW620 cells. i, j, l, o The silencing of mTOR decreased the ECAR & increased the OCR in CT26 & SW620 cells. m The silencing of mTOR decreased the expression of AKT, HIF1 alpha , & proteins encoded by relevant glycolytic genes, such as GLUT1, HK2, LDHA, GPI, PGK1, in CT26 & SW620 cells. p–t Based on TCGA data set analysis, mTOR expression was positively correlated with AKT1, HIF1 alpha , GLUT1, HK2, & LDHA expression. Error bars, mean ± SD, the data are from three independent experiments. Two-sided t tests. *P < 0.05, **P < 0.01, ***P < 0.001, compared with the control group. Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/32313017), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Chromatin Immunoprecipitation: HIF-1 alpha Antibody (H1alpha67) [NB100-105] - HIF-1 alpha -mediated repression of ENT1 expression in Caki-1 cells under hypoxia. Comparison of HIF-1 alpha & HIF-2 alpha protein expression in human matched renal normal-tumor samples (n=11) (A) & 3 RCC cell lines (786-O, 769-P, Caki-1) under normoxia or hypoxia for 6 h (B). (C) Schematic diagram of two hypoxia response elements (HRE) in the promoter of ENT1. (D) CHIP-qPCR analysis of HIF-1 alpha at the promoter of ENT1 in Caki-1 cells, exposed under normoxia or hypoxia for 48 h. Two different HIF-1 alpha antibodies (610958, BD Biosciences; NB100-105, Novus) were used to pull down HIF-1 alpha . The protein expression level of HIF-1 alpha (E) & mRNA expression level of SLC29A1 (F) in Caki-1 under normoxia or hypoxia & HIF-1 alpha knockout-Caki-1 cells. (G) The expression level of ARNT1 (HIF-1 beta ) was detected in Caki-1, transfected with ARNT1 specific siRNAs or NC. Student's t test (two-tailed) was used. (H, I) The expression level of ENT1 in Caki-1, transfected with ARNT1 specific siRNAs or NC, under normoxia or hypoxia. Oneway-ANOVA analysis was used. The results are expressed as mean ± SEM (n=3). ***P<0.001. Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/32206108), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: HIF-1 alpha Antibody (H1alpha67) [NB100-105] - Upregulation of HIF-1 alpha in the spleen restricts CD8+ T cell expansion.(A-B) Mice were infected with 2x107 amastigotes intravenously & euthanized at various time points after infection. HIF-1 alpha expression in splenocytes was assessed by real-time PCR (A) & immunoblot analysis (B). (C-F) 2x104 OT-I CD8+ T cells were adoptively transferred into recipient mice a day prior to infection with ovalbumin-transgenic (PINK) L. donovani amastigotes. (C) Graphs represent the average number of OT-I CD8+ T cells found in the spleen from Hif1aflox/flox & Hif1a+/-mice at d7 & d14 p.i.. (D) Percentage of OT-I CD8+ T cells at d7 & d14 p.i.. (E) Percentage of gated OT-I CD8+ expressing CD62Llo/int & negative for CD127. (F) Percentage of OT-I CD8+ T cells that were CD44+ CD127+CD62Lhi. All data represent mean ± SEM of one of 3 independent experiments, n = 5. * denotes p<0.05 & ** denotes p<0.01. Image collected & cropped by CiteAb from the following publication (//dx.plos.org/10.1371/journal.ppat.1004938), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: HIF-1 alpha Antibody (H1alpha67) [NB100-105] - Effect of 24 hr exposure to fatty acids on expression of proteins involved in energy metabolism.A) Representative western blots, B) Lactate dehydrogenase A (LDHA), C) Phosphoglycerate mutase 1 (PGAM1), D) Hypoxia inducible factor 1 alpha (HIF1 alpha ), E) Isocitrate dehydrogenase (IDH), F) phospho Acetyl CoA carboxylase (ACC)/ACC protein expression. n = 5–6 Protein expression was measured in human bone marrow mesenchymal stem cells (BMMSCs) treated for 24 hr with indicated treatments. The BSA group was treated with media supplemented with 0.55 mM albumin. All fatty acid treated groups were also treated with media supplemented with 0.55 mM albumin in addition to the type & amount of fatty acid indicated in the figures. Values are shown as mean ± SEM. Image collected & cropped by CiteAb from the following publication (//dx.plos.org/10.1371/journal.pone.0120257), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: HIF-1 alpha Antibody (H1alpha67) [NB100-105] - A beta facilitates HIF1 alpha synthesis & autophagy inhibition via mTOR activation. (A) SK-N-MC cells exposed to A beta (5 μM) for 0–48 h. HIF1 alpha & beta -actin expression analyzed by WB. n = 3. (B) Cells pretreated w/ NAC (1 mM) for 30 min prior to A beta treatment for 24 h. HIF1 alpha & beta -actin expression analyzed by WB. n = 3. (C,E) Cells incubated w/ rapamycin (10 nM) for 30 min prior to A beta treatment for 24 h. Phosphorylation of 4EBP1 (Thr 37/46) & 4EBP1, phosphorylation of p70S6K1 (Thr 389), HIF1 alpha & beta -actin analyzed by WB. n = 6. (D) Protein samples immunoprecipitated by eukaryotic translation initiation factor 4E (eIF4E) antibody-conjugated protein A/G agarose beads. Samples blotted w/ 4EBP1 & eIF4E-specific antibodies. n = 3. (F) Cells exposed to PF4708671 (10 μM) for 30 min prior to A beta treatment for 24 h. HIF1 alpha & beta -actin expression detected by WB. n = 6. (G) Cells exposed to cycloheximide (4 μM) for 30 min prior to A beta treatment for 24 h. HIF1 alpha & beta -actin expressions detected by WB. n = 6. (H) Cells pretreated w/ rapamycin (10 nM) for 30 min, incubated w/ A beta for 24 h & analyzed by WB w/ LC3, p62 & beta -actin specific antibodies. n = 3–6. (I) LC3 puncta visualized by confocal microscopy. Presented results merged images. Green & red fluorescents indicate LC3 & PI respectively. Scale bars, 50 μm (magnification × 600). (J) Cells pretreated w/ trehalose (10 μM) for 30 min prior to A beta treatment for 24 h. Cytotoxicity measured by MTT assay at an absorbance of 545 nm using a microplate reader. Data present the mean ± SE. n = 6. (K) Cell viability measured by trypan blue exclusion assay. Data presented as a mean ± SE. n = 6. Each blot image presented as representative image. *p < 0.05 vs. control, #p < 0.05 vs. A beta treatment. Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/28790888), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: HIF-1 alpha Antibody (H1alpha67) [NB100-105] - Ccl7 is a HIF1 alpha direct target gene.a qPCR analysis of Ccl7 mRNA expression in Hif1afl/fl & Hif1a delta SMC VSMCs treated with vehicle, CoCl2, normoxia, or hypoxia (2% O2) for 6, 12, & 24 h. *P < 0.05, **P < 0.01, ***P < 0.001, n = 6 (independent experiments) per group. VSMCs isolated from Hif1afl/fl mice & Hif1a delta SMC mice were infected with oxygen-stable HIF1 alpha -expressing lentivirus, & then treated with Ang II for 24 h, bCcl7 mRNA was measured by qPCR & c CCL7 protein was detected by ELISA. d qPCR analysis of Ccl7 mRNA expression in vehicle or Ang II-treated Hif2afl/fl & Hif2a delta SMC VSMCs. e Schematic diagram of the mouse Ccl7 promoter illustrating the HREs in the regulatory region; the upstream regions were numbered in relation to the transcription initiation site. f Luciferase-reporter constructs under the control of the mouse Ccl7 promoter. HEK293T human embryonic kidney cells transiently transfected with the luciferase construct, & cotransfected with empty vector or HIF1a expression plasmids. Standard dual-luciferase assays were performed. EV, empty vector. **P < 0.01, n = 3. g, h ChIP assays of vehicle or Ang II-treated wild-type VSMCs using HIF1 alpha or HIF2 alpha antibodies. Data were normalized to input. *P < 0.05, **P < 0.01, n = 6 per group. i, j ChIP assays of vehicle or Ang II-treated Hif1afl/fl & Hif1a delta SMC VSMCs using HIF1 alpha antibody. Data were normalized to input. *P < 0.05, **P < 0.01, n = 6 per group. Statistical significance was determined by one-way ANOVA test followed by the unpaired t-test Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/31320613), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: HIF-1 alpha Antibody (H1alpha67) [NB100-105] - HIF1 alpha drives the expression of PFKFB3 in beta -cells. a Luciferase assay showing the activation of PFKFB3 promoter containing 2 hypoxia elements. INS 832/13 overexpressing LacZ or hIAPP were transfected for 36 h with plasmid vectors containing: RenSP luciferase gene without a promoter (empty vector—EV) measuring the background signal or housekeeping gene promoter driving the expression of RenSP luciferase gene ( beta -actin) or PFKFB3 promoter with hypoxia elements driving the expression of RenSP luciferase gene (PFKFB3). b Representative Western blot of HIF1 alpha , PFKFB3, LDHA, Cdh1 in whole cell extracts of INS 832/13 overexpressing LacZ (CTRL) or hIAPP, silenced or not with short hairpin RNA for PFKFB3 (PFKFB3 shRNA) & HIF1 alpha (HIF1 alpha shRNA) for 36 h. CTRL shRNA is non-target shRNA control vector. beta -actin & GAPDH were used as loading control. Data are presented as mean ± SEM, n = 9 independent biological samples (in (a)). Statistical significance was analyzed by unpaired t-test (*p < 0.05) Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/31213603), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Immunocytochemistry/ Immunofluorescence: HIF-1 alpha Antibody (H1alpha67) [NB100-105] - CAPE inhibits MCT-induced HIF-1 alpha expression & PDGF-BB production in rats. (A) Rats were treated with PBS or CAPE (5 or 10 mg/kg) from days 14–28 after MCT treatment 14 days (60 mg/kg). Preparation of lung tissues was analyzed by Western blot to determine the levels of HIF-1 alpha protein. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH)was used as a loading control. (B) Serum levels of PDGF-BB in rat was assayed by an ELISA kit. (C) Representative images of immunohistochemical staining for HIF-1 alpha in lung sections from the rats. Scale bars, 50 μm. (D) Immunofluorescence staining of alpha-smooth muscle actin ( alpha -SMA) & HIF-1 alpha in lung sections from rats. Scale bars, 50 μm. Data in (A,B) are expressed as mean ± SEM of three independent experiments. *** p < 0.01, as compared with the PBS group. ### p < 0.01, as compared with the rats exposed to MCT alone. Results are representative of three rats per experimental group. Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/30909527), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

$Western Blot: HIF-1 alpha Antibody (H1alpha67) [NB100-105] - Role of HIF-1 alpha , FOXO3 in hypoxia-induced BNIP3 expression. (A) UCB-hMSCs were incubated with various durations of hypoxia (0–48 h). The protein expressions of HIF-1 alpha & beta -actin were detected by western blot. n = 4. (B) UCB-hMSCs were pretreated with NAC (5 mM) for 30 min prior to hypoxia incubation for 24 h. The protein expressions of HIF-1 alpha , lamin A/C & beta -tubulin in non-nuclear & nuclear fractionized cell samples were assessed by using western blot. n = 3. (C) UCB-hMSCs were immuno-stained with HIF-1 alpha & PI (magnification ×600). Scale bars, 37.5 µm. (D) HIF1A siRNA or NT siRNA was transfected to cells prior to hypoxia treatment for 24 h. The mRNA expression of BNIP3 was analyzed by qPCR. n = 6. (E) The protein expressions of BNIP3 & HIF-1 alpha were detected by western blot. n = 4. (F) NAC (5 mM) was pretreated to UCB-hMSCs prior to hypoxia treatment for 24 h. FOXO3, lamin A/C & beta -tubulin proteins expressions were assessed by western blot. n = 3. (G) FOXO3 siRNA transfected to UCB-hMSCs prior to hypoxia treatment for 24 h. The FOXO3 mRNA expression was measured by qPCR. n = 6. (H) BNIP3, FOXO3 & beta -actin expressions were detected by western blot. n = 3. Western blot data were normalized by beta -actin, & qPCR data were normalized by ACTB mRNA expression level. Lamin A/C & beta -tubulin were used as nuclear & non-nuclear protein controls, respectively. Quantitative data are presented as a mean ± S.E.M. All blots & confocal images are representative. *p < 0.05 versus control, #p < 0.05 versus hypoxia. Image collected & cropped by CiteAb from the following publication (//linkinghub.elsevier.com/retrieve/pii/S2213231717303804), licensed under a CC-BY license. Not internally tested by Novus Biologicals.$

Western Blot: HIF-1 alpha Antibody (H1alpha67) [NB100-105] - Cx43-knockdown cells regulate the expression of HIF-1 alpha , c-Src, VEGF & the proliferation of endothelial cells. (a) The expression of Cx43, c-Src, & HIF-1 alpha was measured in Cx43-knocknown cells by Western blot analysis. beta -actin expression served as loading controls for & total protein. Inserted values indicated relative protein expression in comparison with beta -actin; (b) Cx43-knockdown cells were cotransfected with pCLNCX-6× HRELuc & pTCYLacZ plasmids. Then, cells were treated with 100 ng/μL PP2 or DMSO for 1 h. At 6 h post-transfection, their luciferase activities were determined & normalized with beta -gal activity. Data shown were the mean ± SD (n = 4); (c) The conditioned medium of Cx43-knockdown cells was measured by ELISA. Data shown were the mean ± SD (n = 4); (d) The conditioned medium of Cx43-knockdown cells reduced the proliferation of endothelial cells. The HEMC-1 cells treated with conditioned medium of Cx43-knockdown cells were examined for proliferation activity. Cell viability was assessed by the WST-1 assay. Data shown were the mean ± SD (n = 4). * p < 0.05; ** p < 0.01; *** p < 0.001. Image collected & cropped by CiteAb from the following publication (//www.mdpi.com/1422-0067/16/1/439), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: HIF-1 alpha Antibody (H1alpha67) [NB100-105] - Involvement of CBP in BNIP3 expression regulated by HIF-1 alpha & FOXO3 under hypoxia. (A) UCB-hMSCs were incubated with hypoxia condition for 24 h. Co-immunoprecipitation of HIF-1 alpha & FOXO3 with IgG & CBP were shown in left panel. IgG was used as a negative control. The total protein expressions of HIF-1 alpha , FOXO3, CBP & beta -actin in lysate were shown in right panel. n = 3. (B) CBP (20 μM) was pretreated to UCB-hMSCs, & cells were incubated with hypoxia for 24 h. The BNIP3 mRNA expression level was analyzed by qPCR. n = 6. (C) BNIP3 & beta -actin protein expressions were analyzed by western blot. Data represent mean ± S.E. n = 4. (D, E) Sample DNA was immuno-precipitated with RNA polymerase, IgG, HIF-1 alpha & FOXO3 specific antibodies. CHIP (top panel) & lysate (bottom panel) samples were amplified with the primers of GAPDH & BNIP3 promoters. Quantitative CHIP data was analyzed by qPCR, & shown in the right panel. n = 4. Western blot data were normalized by beta -actin, & qPCR data were normalized by ACTB mRNA expression level. Quantitative data are presented as a mean ± S.E.M. All blot images are representative. *p < 0.05 versus control, #p < 0.05 versus hypoxia. Image collected & cropped by CiteAb from the following publication (//linkinghub.elsevier.com/retrieve/pii/S2213231717303804), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Immunohistochemistry: HIF-1 alpha Antibody (H1alpha67) [NB100-105] - Precision quantitative image analysis workflow for the characterization of functional ITH.(a) Representative areas with positive immunohistochemical (IHC) staining for HIF-1 alpha , HIF-2 alpha , phospho-mTOR S2448, phospho-S6RP S235/236 & Ki-67. Only nuclear HIF-1 alpha & HIF-2 alpha expression was included in the analysis. Scale bar, 100 μm. (b) Overview of the quantitative IHC analysis process using a representative image of a whole-slide scanned pT1M1 tumour after phospho-mTOR S2448 staining. The image was overlaid with a virtual grid consisting of 1 mm2 squares that defines the tumour regions to be analysed. Non-cancerous tissue was subtracted manually (dark grey) & a positive pixel count was performed on the corrected area. Positivity per square was expressed as percentage of positive pixels. If a square contained non-tumorous tissue, the positivity was normalized to the corrected square area. Computed values were depicted as a heat map. Coloured squares represent tumour, whereas open squares indicate completely non-tumorous areas. Squares defined as peripheral tumour zone are marked by a black line. Scale bar, 1 cm. Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/27291893), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: HIF-1 alpha Antibody (H1alpha67) [NB100-105] - Glucose uptake & mitochondrial clustering following RT & CT. Glucose uptake (a,b) & mitochondrial clustering (c,d) was evaluated in A549 & A549RR cells before, 12 & 24 hours following radiation alone (RT) or combined radiation & YC-1 treatment. Immunoblotting was performed to verify HIF-1 alpha & PDK-1 inhibition (e–g). Sample immunoblots were cropped horizontally at indicated molecular weight. Uncropped exposures can be seen in supplemental Fig. 1. Significant differences (p < 0.05) are indicated by a bar starting & ending over groups that are different. Dashed line extends across at the mean of the A549 control prior to RT & without YC-1. RT – Radiation Treatment; CT – Combination treatment; ME – Main effect only with no significant interactions present; NS – No significant differences. Scale bar in images represents 50 μm. Image collected & cropped by CiteAb from the following publication (//www.nature.com/articles/s41598-018-27262-y), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Immunocytochemistry/ Immunofluorescence: HIF-1 alpha Antibody (H1alpha67) [NB100-105] - Regulation of OA development by HIF-1 alpha . Plain radiographs of the entire bodies at 8 weeks of age (A), & knee joints 8 weeks after OA surgery (B) of Cntl & cKO littermates. Scale bars, 10 mm & 1 mm, respectively. (C) Safranin O & TUNEL staining’s of Cntl & KO knee joints at 8 weeks after OA surgery (n = 9 & 11, respectively). Tamoxifen induction was performed at 7 weeks. Inset boxes in top images indicate the regions of high magnification Safranin O & TUNEL images. Inset boxes in middle images indicate the regions of immunofluorescence in (G). White dotted lines in TUNEL images indicate the cartilage area. Scale bars, 400 µm & 100 µm, respectively. (D) OARSI (Osteoarthritis Research Society International) score of OA development. Lines show the mean ± SD. *P < 0.05. (E) Rate of TUNEL-positive cells in the cartilage area. Bars show the mean ± SD. *P < 0.05. (F) mRNA levels of Hif1a in articular cartilage tissues of Cntl & KO. Bars show the mean ± SD. *P < 0.05. (G) Immunofluorescence of Hif1a, Mmp13, Hif2a, I kappa B alpha , Ser 32 & 36 dual phosphorylated I kappa B alpha , Rela, & C1qtnf3. Scale bars, 20 µm. The percentage of positive cells in the immunofluorescence is shown below. *P < 0.05. Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/32214220), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: HIF-1 alpha Antibody (H1alpha67) [NB100-105] - TSA reduces HIF-1 alpha & VEGF expression & up-regulates the expression of PEDF.Real-time PCR (A) & Western blot assays (B) were performed on RPE cells treated with 0–0.5 μM TSA & 150 μM CoCl2. (A) Cells were treated with 0–0.5 μM TSA for 14 h & then co-treated with 150 μM CoCl2 for 6 h for the analysis of gene expression by real-time PCR. Changes in HIF-1 alpha mRNA levels were not statistically significant. CoCl2 causes a fourfold enhancement of VEGF mRNA expression; but at 0.5 μM TSA, the mRNA level of VEGF reduces to less than half of that in cells treated with CoCl2 only. TSA induces a statistically significant increase in the mRNA level of PEDF. (B) Cells were treated with 0–0.5 μM TSA for 18 h & then co-treated with 150 μM CoCl2 for 6 h for Western blot analysis. TSA reduces the CoCl2-induced HIF-1 alpha & VEGF protein levels by 4.3-fold & 5.7-fold, respectively, & up-regulates PEDF protein level by threefold. (C) Densitometry data for Western blot of HIF-1 alpha , VEGF & PEDF. (*: t test p<0.05; **: t test p<0.01). Image collected & cropped by CiteAb from the following publication (//dx.plos.org/10.1371/journal.pone.0120587), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

$Western Blot: HIF-1 alpha Antibody (H1alpha67) [NB100-105] - Role of HIF-1 alpha , FOXO3 in hypoxia-induced BNIP3 expression. (A) UCB-hMSCs were incubated with various durations of hypoxia (0–48 h). The protein expressions of HIF-1 alpha & beta -actin were detected by western blot. n = 4. (B) UCB-hMSCs were pretreated with NAC (5 mM) for 30 min prior to hypoxia incubation for 24 h. The protein expressions of HIF-1 alpha , lamin A/C & beta -tubulin in non-nuclear & nuclear fractionized cell samples were assessed by using western blot. n = 3. (C) UCB-hMSCs were immuno-stained with HIF-1 alpha & PI (magnification ×600). Scale bars, 37.5 µm. (D) HIF1A siRNA or NT siRNA was transfected to cells prior to hypoxia treatment for 24 h. The mRNA expression of BNIP3 was analyzed by qPCR. n = 6. (E) The protein expressions of BNIP3 & HIF-1 alpha were detected by western blot. n = 4. (F) NAC (5 mM) was pretreated to UCB-hMSCs prior to hypoxia treatment for 24 h. FOXO3, lamin A/C & beta -tubulin proteins expressions were assessed by western blot. n = 3. (G) FOXO3 siRNA transfected to UCB-hMSCs prior to hypoxia treatment for 24 h. The FOXO3 mRNA expression was measured by qPCR. n = 6. (H) BNIP3, FOXO3 & beta -actin expressions were detected by western blot. n = 3. Western blot data were normalized by beta -actin, & qPCR data were normalized by ACTB mRNA expression level. Lamin A/C & beta -tubulin were used as nuclear & non-nuclear protein controls, respectively. Quantitative data are presented as a mean ± S.E.M. All blots & confocal images are representative. *p < 0.05 versus control, #p < 0.05 versus hypoxia. Image collected & cropped by CiteAb from the following publication (//linkinghub.elsevier.com/retrieve/pii/S2213231717303804), licensed under a CC-BY license. Not internally tested by Novus Biologicals.$

Western Blot: HIF-1 alpha Antibody (H1alpha67) [NB100-105] - HIF1 alpha protein is suppressed by ED71 but not by 1,25(OH)2D3.(A) Western analysis of Raw264.7 cells cultured in hypoxic conditions with or without 10−7 M of ED71 or 1,25(OH)2D3 (1,25D). (B) Hif1 alpha mRNA levels in Raw264.7 cells cultured in hypoxic conditions were analyzed by realtime PCR in the presence or absence of 10−7 M ED71 or 1,25(OH)2D3. Data represent mean Hif1 alpha expression relative to that of Actb ± SD (n = 5). (C) Levels of VDR transcripts in Raw264.7 cells transfected with shRNA targeting the VDR (shVDR) or control shRNA (Control) were determined by realtime PCR. Data represent mean VDR expression relative to that of Actb ± SD (n = 5). (D) Western analysis of control (shControl) or VDR-suppressed (shVDR#1 or shVDR#2) Raw264.7 transformants cultured in hypoxic conditions with ED71 or 1,25(OH)2D3 (1,25D), both at 10−7 M. (E) M-CSF-dependent Ctsk Cre/Hifflox/flox cells were cultured in normoxic conditions to suppress HIF1 alpha in the presence of M-CSF (50 ng/ml) plus RANKL (25 ng/ml) with either ED71 or 1,25(OH)2D3 (1,25D) both at 10−7M for 4 days. Expression of Ctsk & NFATc1 was then assessed by realtime PCR. Data represent mean Ctsk or NFATc1 expression relative to that of Actb ± SD (n = 5). *P<0.05; **P<0.01; ***P<0.001. Image collected & cropped by CiteAb from the following publication (//dx.plos.org/10.1371/journal.pone.0111845), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: HIF-1 alpha Antibody (H1alpha67) [NB100-105] - Effect of PACAP, PACAP6-38, VIP, & VIP antagonist on expression of HIF-1 alpha , HIF-2 alpha , & EGFR in U87-MG cells under normoxia & hypoxia. (A) Representative immunoblots of HIF-1 alpha , HIF-2 alpha , & EGFR expression on U87MG cells grown normoxia or exposed to hypoxia. (B) The bar graphs show quantitative analysis of signals obtained by immunoblots resulting from three independent experiments. Relative band densities were quantified by using ImageJ software. Protein levels are expressed as arbitrary units obtained after normalization to beta -tubulin which was used as loading control. Data represent means ± SEM (*p < 0.05, **p < 0.01, or ***p < 0.001 vs. Vhl under normoxia; ###p < 0.001 vs. Vhl under hypoxia $$p < 0.01 or $$$p < 0.001 vs. PACAP, as determined by one-way ANOVA followed by the Tukey post hoc test). Image collected & cropped by CiteAb from the following publication (//journal.frontiersin.org/Article/10.3389/fphar.2016.00139/abstract), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: HIF-1 alpha Antibody (H1alpha67) [NB100-105] - Lack of HIF-P4H-1 increases the amount of p53. (a) Western blot analysis of p53 in wt & Hif-p4h-1−/− MEFs cultured in 21% or 1% O2 for 24 h. (b,c) Western blot analysis of p53 in Hif-p4h-1 & scrambled (Scrm) siRNA transfected HEK293 cells (b) & wt MEFs treated with 50 µM FG4497 for 24 h (c). (d) qPCR analysis of p53 mRNA in wt & Hif-p4h-1−/− MEFs cultured in 21% or 1% O2 for 24 h. (e) Analysis of p53 protein turnover rate. Hif-p4h-1−/− & wt MEFs were treated with 200 μg/ml of cycloheximide for the indicated time points & whole cell lysates were blotted for p53. (f) Western blot analysis of MDM2 in wt & Hif-p4h-1−/− MEFs cultured in 21% or 1% O2 for 24 h. (g) Western blot analysis of p53 & MDM2 in wt MEFs treated with 10 μM nutlin-3a for 24 h with or without overexpression of human HIF-P4H-1 (OE). (h,i) Western blot analysis of HIF1 alpha in wt & Hif-p4h-1−/− MEFs cultured in 21% or 1% O2 for 24 h (h) & in scrambled & Hif-p4h-1 siRNA transfected HEK293 cells (i). (j) Western blot analysis of ubiquitination of p53 in Hif-p4h-1 & scrambled siRNA transfected HEK293 cells. The cells were transfected with HA-ubiquitin & endogenous p53 was immunoprecipitated followed by Western blotting with anti-HA & anti-P53 antibodies. (k) Western blot analysis of p53 in Hif-p4h-1−/− & wt MEFs were treated with or without 10 μM MG132 for 5 h. Data are presented as representative Western blots & as mean ± s.d., n = at least 3 individual MEF isolates or experiments. *P < 0.05, **P < 0.01 & ***P < 0.001, two-tailed Student’s t-test. Unprocessed original scans of blots are shown in Supplementary Fig. 5. Image collected & cropped by CiteAb from the following publication (//www.nature.com/articles/s41598-017-17376-0), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: HIF-1 alpha Antibody (H1alpha67) [NB100-105] - NDRG1 was regulated by HIF‐1/2 alpha . (A‐E) The protein expression of the shown genes was detected by Western blot, respectively; beta ‐actin was used as the loading control. (A) RCC4 & 786‐O cells (B) were infected with VHL vector (VHL) or empty vector (EV). (C) Caki‐1 cells were transfected with VHL shRNAs (shVHL) or non‐specific control (NC). (D) RCC4 cells were infected with HIF‐1 alpha siRNAs (siHIF‐1 alpha ) or non‐specific control (NC). (E) 786‐O cells were transfected with HIF‐2 alpha shRNAs (shHIF‐2 alpha ) or non‐specific control (NC). (F) Representative images of HIF‐1 alpha , HIF‐2 alpha & NDRG1 protein expression from the IHC staining of human clear cell renal cell cancer specimens. Patient I: The protein levels of HIF‐1 alpha , HIF‐2 alpha & NDRG1 were low. Patient II: The protein levels of HIF‐1 alpha , HIF‐2 alpha & NDRG1 were medium. Patient III: The protein levels of HIF‐1 alpha , HIF‐2 alpha & NDRG1 were high (scale bar, 50 μm). (G & H) The protein level of NDRG1 is significantly correlated with HIF‐1 alpha (G) & HIF‐2 alpha (H). The protein levels were evaluated according to IHC scores, representing very low (score 0‐2), low (score 3‐4), high (score 5‐8) & strong (score 9‐12). The subjects were divided into four groups according to the IHC scores of HIFs in the tumours. (I) The protein level of NDRG1 is significantly correlated with the average of HIF‐1 alpha & HIF‐2 alpha in ccRCC tissues. The protein levels are evaluated according to IHC scores, representing very low (score < 3), low (score < 5), high (score < 8) & strong (score 8‐12). The subjects were divided into four groups according to the IHC scores of the average of HIF‐1 alpha & HIF‐2 alpha . Significance p‐values & r values according to Spearman's rank correlation Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/32537867), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: HIF-1 alpha Antibody (H1alpha67) [NB100-105] - In-vivo lentiviral delivery of Cre-recombinase to renal tubular epithelium results in recombination of target genes.(a) Diagram of pCCIE lentiviral construct. Cre, Cre-recombinase; IRES, internal ribosome entry site; GFP, Green fluorescent protein. (b) Anti-VHL, HIF1a & GAPDH immunoblots of renal cortical protein lysates from Vhlwt/wt & Vhlfl/fl mice intrarenally injected with CCIE. Samples were collected 12 months post infection with each column representing an individual mouse. Blots were cropped to improve clarity, full-length blots are presented in Supplementary Fig. S3a. (c) Histological images of renal sections from Vhlwt/wt & Vhlfl/fl mice intrarenally injected with CCIE at 12 months post injection (stains & antibodies as indicated). Scale bars, 100 μm. Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/26046460), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: HIF-1 alpha Antibody (H1alpha67) [NB100-105] - Induction of HIF-1 alpha & a glycolytic response by VLX600.(a) GSEA plots of hypoxia, glycolysis, mitosis & p53 networks based on the gene expression profiles of HCT116 MCS or HCT116HIF-1 alpha −/− MCS exposed for 6 h to 6 μM VLX600. NES, normalized enrichment score; P values refer to the nominal P values generated after 1,000 permutations. (b) Monolayer HCT116 cells were exposed to 6 μM VLX600 & analysed for HIF-1 alpha expression by western blotting after 6 h. (c) Spheroids were exposed to VLX600 for different times, sectioned & stained for HIF-1 alpha . The spheroid core areas were lack of HIF-1 alpha induction. Scale bar, 250 μm (d) HCT116HIF-1 alpha −/− MCS show strong positivity for active caspase-3 in core areas. Spheroids were fixed & stained for active caspase-3 5 days after formation; scale bar, 250 μm. (e) HCT116 & HCT116HIF-1 alpha −/− MCS were exposed for 72 h to VLX600 (6 μM) followed by drug wash-out. Diameters of the MCS were recorded & converted to volumes. Volume determinations are inaccurate at 4–10 days since MCS disintegrate & dead cells stick to spheroid structures (the transient increase in the volume of VLX600-treated HCT116HIF-1 alpha −/− MCS is likely to be an artifact). Shown are means±s.d. (n=3). (f) HCT116, HCT116HIF-1 alpha −/− or HCT116p53−/− monolayer cells were exposed to VLX600 & viability was determined after 72 h using the fluorometric microculture cytotoxicity assay. Shown are means±s.e.m. (n=3). (g) Monolayer HCT116 cells were exposed to 6 μM VLX600 & analysed for p53 expression by immunoblotting at the times indicated. Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/24548894), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Reactivity:

Hu, Mu, Rt, Po, Bv, Ca, Fe, Ma, Pm, Pm, Rb, Sh, Xp

Species Glossary

Reactivity: Hu, Mu, Rt, Po, Bv, Ca, Fe, Ma, Pm, Pm, Rb, Sh, Xp

Applications:

, , , , , , , , , , , , , , ,

Applications: , , , , , , , , , , , , , , ,

Host:

Mouse Monoclonal

Host: Mouse Monoclonal

Sizes:

Sample Size Available

Sizes: Sample Size Available

Conjugate:

Unconjugated

Available Conjugates:

Validated by:

Validated by:

Conjugate	Catalog #	Availability	Size	Price
Alexa Fluor 350	NB100-105AF350
Alexa Fluor 405	NB100-105AF405
Alexa Fluor 488	NB100-105AF488
Alexa Fluor 532	NB100-105AF532
Alexa Fluor 594	NB100-105AF594
Alexa Fluor 647	NB100-105AF647
Alexa Fluor 700	NB100-105AF700
Alexa Fluor 750	NB100-105AF750
Allophycocyanin	NB100-105APC
Biotin	NB100-105B
CoraFluor 1	NB100-105CL1
DyLight 350	NB100-105UV
DyLight 405	NB100-105V
DyLight 488	NB100-105G
DyLight 550	NB100-105R
DyLight 594	NB100-105DL594
DyLight 650	NB100-105C
DyLight 680	NB100-105FR
DyLight 755	NB100-105IR
FITC	NB100-105F
HRP	NB100-105H
Janelia Fluor 525	NB100-105JF525
Janelia Fluor 549	NB100-105JF549
Janelia Fluor 646	NB100-105JF646
Janelia Fluor 669	NB100-105JF669
PE	NB100-105PE
PerCP	NB100-105PCP
mFluor Violet 450 SE	NB100-105MFV450
mFluor Violet 500 SE	NB100-105MFV500
mFluor Violet 610 SE	NB100-105MFV610

Available Formulations

Formulation	Catalog #	Availability	Price
Azide and BSA Free	NBP2-80760

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NB100-122

HIF-2 alpha/EPAS1 Antibody - BSA Free

(37 Reviews)

(826 Publications)

0.1 ml

NB100-122

WNT11 is induced by hypoxia or hypoxic mimetics in different cell types. Immunoblot analyses of HeLa cells under normal air or hypoxia for 24 hrs. Image collected and cropped by CiteAb from the following publication (//www.nature.com/articles/srep21520) licensed under a CC-BY license.

<b>Biological Strategies Validation. </b>HIF-1alpha is the predominant transcriptional regulator of WNT11 expression during hypoxia. EMSCs isolated from the indicated mouse genotypes were infected with lentivirus expressing GFP or Cre recombinase. Non-infected cells and GFP infected cells served as controls. Immunoblot analyses of EMSCs derived from the indicated genotypes treated with 0.1 mM DMOG for 24 hrs. Attenuated WNT11 expression in Hif-1alpha KO EMSCs (lenti-Cre infected Hif-1af/f). Image collected and cropped by CiteAb from the following publication (//www.nature.com/articles/srep21520) licensed under a CC-BY license.

<b>Orthogonal Strategies Validation. </b>Formalin-fixed paraffin-embedded tissue sections of human placenta were probed for HIF-2 alpha/EPAS1 mRNA (ACD RNAScope Probe, catalog #410598; Fast Red chromogen, ACD catalog # 322750). Adjacent tissue section was processed for immunohistochemistry using Rabbit Polyclonal (Novus Biologicals catalog # NB100-122) at 1:100 dilution with one-hour incubation at room temperature followed by incubation with anti-rabbit IgG VisUCyte HRP Polymer Antibody (Catalog # VC003) and DAB chromogen (yellow-brown). Tissue was counterstained with hematoxylin (blue). Specific staining was localized to trophoblastic cells.

Expression of hypoxia-inducible alpha subunits in normal and diseased lung tissue. HIF-2alpha expression are more evident in fibroblasts from idiopathic pulmonary fibrosis (d) than from lung tissue affected by other inflammatory conditions (i.e. chronic bronchitis, panel e) or normal lung tissue (f); as demonstrated by a higher proportion of positive fibroblasts (open arrows) than negative ones (solid black arrows). Image collected and cropped by CiteAb from the following publication (//respiratory-research.biomedcentral.com/articles/10.1186/s12931-019-1100-4) licensed under a CC-BY license.

Image shows a specific band for HIF-2 alpha in 0.5 mg/mL of hypoxic HeLa lysate. This experiment was performed under reducing conditions using the 12-230 kDa separation system.

<b>Genetic Strategies Validation. </b>Immunoblot validation of HIF2A and PHD2 KO clones using HIF2A (#NB100-122; dilution: 1/300), PHD2 (#NB100-137; dilution: 1/500). To blot HIFs factor cells were first pre-treated for 5 h with CoCl2 300 uM before protein extraction, a condition that promotes HIF factor accumulation. Image collected and cropped by CiteAb from the following publication (//www.nature.com/articles/s41467-018-06988-3) licensed under a CC-BY license.

<b>Biological Strategies Validation. </b>HIF-2 alpha in human retinal and choroidal primary endothelia lysates using .. Image from verified customer review.

<b>Biological Strategies Validation. </b>HIF-2 alpha in MDA-MB-231 cell lysate (overexpression and endogenous samples) using . did not react to HIF-1 alpha overexpression. Image from verified customer review.

Induced WNT11 expression with tumor hypoxia and WNT11 regulates tumor growth. Bevacizumab increased expression of HIF-1alpha and HIF-2alpha and WNT11. First 10 lanes are control tumors, and the last 10 lanes are tumors from bevacizumab-treated animals. Lysates from whole tissue and nuclei are indicated. Alpha -Tubulin, actin and lamin A/C are loading controls. Image collected and cropped by CiteAb from the following publication (//www.nature.com/articles/srep21520) licensed under a CC-BY license.

MDA-MB-231 cells were exposed to 20% or 1% O2 for 16 hours, and chromatin immunoprecipitation (ChIP) was performed with the indicated antibody (Ab). Primers flanking the HIF binding site were used for qPCR. ChIP image submitted by a verified customer review.

<b>Biological Strategies Validation. </b>Lane 1: Cobalt chloride treated COS7 nuclear extracts. Lane 2: Untreated COS7 nuclear extracts.

786-O cells without or with VHL overexpression. Image from verified customer review.

<b>Biological Strategies Validation. </b>Analysis using the HRP conjugate of NB100-122. Detection of normoxic and hypoxic nuclear rat cell lysates.

HIF-2 alpha immunoreactivity in human cardiac myocytes stained with NB100-122.

Expression and correlation of 3 antibodies: HIF-1 alpha, HIF-2 alpha and HAF in ccRCC. (A, C & E) Positive nuclear staining to the 3 antibodies: HIF-1 alpha, HIF-2 alpha and HAF, in primary ccRCCs at high magnification (100X). (B, D & F) Heterogenous nuclear staining of 3 antibodies in a tumor at low magnification (20X). (G, H and I). Correlation of 3 antibodies with the Fuhrman grade.Citation: Ambrosetti D, Dufies M, Dadone B, Durand M, Borchiellini D, Amiel J, et al. (2018) The two glycolytic markers GLUT1 and MCT1 correlate with tumor grade and survival in clear-cell renal cell carcinoma. PLoS ONE 13(2): e0193477. //doi.org/10.1371/journal.pone.0193477

<b>Genetic Strategies Validation. </b>Reduction of HIF-2alpha levels leads to protection in UV-triggered apoptosis, but not for apoptosis caused by glucose and serum starvation in 786-O cells. Parental 786-O or those either stably expressing wild-type VHLp19 or stably infected with a control vector (pSuperRetro) or a pool of two HIF-2alpha shRNAs vectors [21] were grown to confluence and lysed. Cell alpha-tubulin.

Sp1 and HIFs contribute to the synergistic activation of multiple genes in OVSAYO cells under SSH.b) Activation of multiple genes under hypoxia is dependent on HIFs. Western blotting is also shown for HIFs. Data shown are the mean (n = 3) ± SD.

Western Blot: Rabbit Polyclonal HIF-2 alpha/EPAS1 Antibody [NB100-122] - Analysis of HIF-2 alpha/EPAS1 antibody on mouse liver nuclear extracts. Image from a verified customer review.

Immunocytochemistry/ Immunofluorescence: HIF-2 alpha/EPAS1 Antibody - BSA Free [NB100-122] - Muscle repair following eccentric contraction–induced injury is concomitant with dynamic alterations of HIF2A & HIF1A expression in SCs.(A–C) Representative images of EDL myofibers from injured muscles at various time points (n >50 myofibers from 3 mice/group/time point) & stained for Pax7, DAPI, & EdU (A), HIF2A (B), or HIF1A (C). Scale bars: 20 μm. Arrowheads indicate SCs. (D) Number of Pax7+ SCs per myofiber at various time points. (E) Percentage of EdU+ SCs at various time points. (F) Percentage of HIF2A+ SCs at various time points. (G) Percentage of HIF1A+ SCs at various time points. Data represent the mean ± SEM. Image collected & cropped by CiteAb from the following publication (//www.jci.org/articles/view/96208), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Immunocytochemistry/ Immunofluorescence: HIF-2 alpha/EPAS1 Antibody - BSA Free [NB100-122] - Genetic ablation of HIF2A in QSCs leads to transient activation, proliferation, & differentiation of SCs.(B) Representative images of myofibers from SC-HIF2AKO mice & control littermates (n >50 myofibers from 5 mice/group; 10 dpr). Immunofluorescence of Pax7 (red), HIF2A (green), MyoD (purple), & DAPI (blue) staining revealed HIF2A–MyoD+ & HIF2A+MyoD– SCs (arrowheads) in SC-HIF2AKO & control mice, respectively. Scale bar: 10 μm. Image collected & cropped by CiteAb from following publication (//www.jci.org/articles/view/96208), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Immunocytochemistry/ Immunofluorescence: HIF-2 alpha/EPAS1 Antibody - BSA Free [NB100-122] - EpEX & EpCAM activate a STAT3-HIF2 alpha signal for EpEX/EpCAM-mediated iPSC formation.(A) iPSCs were infected with two different EpCAM shRNAs (two clones, #1 & #2). The protein expressions of EpCAM & HIF2 alpha were detected by Western blotting. (B) MEFs were stimulated by EpEX (1 μg/mL) at the indicated times. Nuclear-translocation was detected with a specific antibody against HIF2 alpha (n = 3). (C) Immunofluorescence staining was performed to detect subcellular localization of HIF2 alpha . Nuclei were stained with DAPI. Scale bar: 10 μm. (D) MEFs were treated with STAT3 inhibitor (WP1066, 10 μM), & then stimulated with EpEX for 30 min. The nuclear-translocation of HIF2 alpha was detected by Western blotting with anti-HIF2 alpha antibody (n = 3). (E) iPSC morphology was observed at day 20 after induction. Reprogramming of Oct4-GFP MEFs was induced by transfection of OSKM, OE + EpEX, & KE + EpEX with or without STAT3 inhibitor WP1066, or HIF2 alpha shRNA (n = 3). Scale bar: 50 μm. Image collected & cropped by CiteAb from the following publication (//www.nature.com/articles/srep41852), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Gel Super Shift Assays: HIF-2 alpha/EPAS1 Antibody - BSA Free [NB100-122] - Manipulation of DNA methylation at HRE sequences alters HIF binding. (A) The Hypoxia Response Element (HRE) contains a CpG site that can be methylated which prevents HIF1 & HIF2 binding in vitro. EMSA of in vitro–translated HIF1 & HIF2 binding to 32P-labelled unmethylated & methylated HRE probes. Competition with 250X molar excess of unlabelled/unmethylated HRE probe (lanes 3, 7,12, 16) or unlabelled control HRE-free probe (lanes 4, 8, 13, 17). HIF1 & HIF2 complexes bound to unmethylated HRE or methylated HRE supershifted with anti-HIF1 alpha (lanes 9 & 18) & anti-HIF2 alpha (lanes 5 & 14). (B) RCC4-VHL cells were grown in normoxia (NT) or treated with 25 µM decitabine (5aza) or grown in hypoxia (1%) for 48 hrs & with the addition of 25 µM decitabine (1% + 5aza). QPCR was performed using primers specific to VEGF, uPAR, TGF alpha , GUS, U1AsnRNP1. Samples were normalised to relative expression of the housekeeping gene, ACTIN. Image collected & cropped by CiteAb from the following publication (//www.nature.com/articles/s41598-018-21524-5), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Immunocytochemistry/ Immunofluorescence: HIF-2 alpha/EPAS1 Antibody - BSA Free [NB100-122] - HIF-1 alpha /2 alpha expression in myeloid-specific KO mice targeting the HIF pathway. (A) Images of the colon of wild-type (WT), myeloid-specific Hif-1a KO (hMRP8 Hif-1a KO) or von Hippel Lindau (Vhl) KO (hMRP8 Vhl KO) mice, immunostained for MRP8 (green) & the DNA-binding regions of Hif-1a mRNA (red). Mice were fed with 5% DSS for 4 days prior to immunostaining analyses. Note that there were no MRP8-positive cells that were positive for Hif-1a mRNA in hMRP8 Hif-1a KO (middle column) mice, but we observed many cells that were double positive for MRP8 & Hif-1a mRNA in hMRP8 Vhl KO mice (right column). (B) Images of the colon of hMRP8 Vhl KO mice fed with 5% DSS as in A, immunostained for MRP8 (green) & HIF-1 alpha (red, upper row) or HIF-2 alpha (red, bottom row). DAPI-stained nuclei are shown in blue. White boxes in A & B indicate the regions magnified in the lower or right images, respectively. Yellow arrowheads in A & B indicate cells positive for both markers. Scale bars: 100 μm. Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/29967068), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Immunocytochemistry/ Immunofluorescence: HIF-2 alpha/EPAS1 Antibody - BSA Free [NB100-122] - HIF2 alpha stabilization results in exocrine cell atrophy & expansion of duct-like tubular structures. (A) Body weight (left panel), pancreas weight (middle panel) & body/pancreas weight ratio (right panel) in Pdx1-Cre;HIF2dPA & control mice at 2 & 8 weeks of age. Data are presented as mean ± SD. (B) HIF2 alpha accumulation in Pdx1-Cre;HIF2dPA analyzed by Western blot with anti-HA antibody. Two independent two-week-old control & mutant mice are shown. beta -actin protein was used for loading control. Full-length blots are presented in Supplementary Fig. 2. (D) Immunofluorescence analysis of HIF2 alpha in two-week-old control pancreata. Endogenous HIF2 alpha expression is observed in islets (marked by an white asterisk) but not in exocrine tissue. (D) Robust HIF2 alpha accumulation in the pancreas of two-week-old Pdx1-Cre;HIF2dPA mice. Hematoxylin/Eosin-stained pancreatic sections from P0 (E,F), two- (I,J) & eight-week-old (M,N) Pdx1-Cre;HIF2dPA & control mice. Inset in N shows an area with adipose tissue in Pdx1-Cre;HIF2dPA pancreata. Immunofluorescence of amylase & KRT19 shows no differences between Pdx1-Cre;HIF2dPA & control mice at P0 (G,H). Duct-like tubular structures & loss of amylase immunoreactivity in two- (K,L) & eight-week-old (O,P) Pdx1-Cre;HIF2dPA mice compared to control mice. Note areas with normal acini in 8-week-old Pdx1-Cre;HIF2dPA mice (white asterisk in O). Insets in (H,L & P) show higher magnification pictures. DAPI staining is shown in blue in (C,D,G,H,K,L,O & P). Scale bars = 50 µm for (C,D); 100 µm for (E–P). ***P 50 myofibers from n = 3 mice) showing that nmGFP+ QSCs were pimonidazole+. Scale bars: 50 μm & 10 μm (insets). Inset images show that pimonidazole signals were relatively enriched in the cytoplasm of QSCs. Arrowheads indicate a QSC; asterisks indicate a myonucleus. (B) Percentage of pimonidazole+ QSCs. (C) Timeline of in vivo CCI-103F labeling in C57BL/6 mice & representative images of uninjured/resting EDL myofibers (n >50 myofibers from 3 mice) showing that nmGFP+ QSCs were CCI-103F+. Arrowheads indicate a QSC; asterisks indicate a myonucleus. Scale bar: 20 μm. (D) Percentage of CCI-103F+ QSCs. (E & F) Representative images of uninjured/resting EDL myofibers from C57BL/6 mice (n >50 myofibers from 6 mice/group) showing that most QSCs were HIF2A+, but HIF1A–. Scale bars: 10 μm. (G) Percentage of HIF1A+ & HIF2A+ QSCs. Data represent the mean ± SEM. Image collected & cropped by CiteAb from the following publication (//www.jci.org/articles/view/96208), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Reactivity:

Hu, Mu, Rt, Fi, Ha, Pm, Rb, Re, Sh

Species Glossary

Reactivity: Hu, Mu, Rt, Fi, Ha, Pm, Rb, Re, Sh

Applications:

, , , , , , , , , , , , , ,

Applications: , , , , , , , , , , , , , ,

Host:

Rabbit Polyclonal

Host: Rabbit Polyclonal

Sizes:

Sample Size Available

Sizes: Sample Size Available

Conjugate:

Unconjugated

Available Conjugates:

Validated by:

Validated by:

Conjugate	Catalog #	Availability	Size	Price
Alexa Fluor 350	NB100-122AF350
Alexa Fluor 405	NB100-122AF405
Alexa Fluor 488	NB100-122AF488
Alexa Fluor 532	NB100-122AF532
Alexa Fluor 594	NB100-122AF594
Alexa Fluor 647	NB100-122AF647
Alexa Fluor 700	NB100-122AF700
Alexa Fluor 750	NB100-122AF750
Allophycocyanin	NB100-122APC
Allophycocyanin/Cy7	NB100-122APCCY7
Biotin	NB100-122B
CoraFluor 1	NB100-122CL1
DyLight 350	NB100-122UV
DyLight 405	NB100-122V
DyLight 488	NB100-122G
DyLight 550	NB100-122R
DyLight 594	NB100-122DL594
DyLight 650	NB100-122C
DyLight 680	NB100-122FR
DyLight 755	NB100-122IR
FITC	NB100-122F
HRP	NB100-122H
Janelia Fluor 525	NB100-122JF525
Janelia Fluor 549	NB100-122JF549
Janelia Fluor 646	NB100-122JF646
Janelia Fluor 669	NB100-122JF669
PE	NB100-122PE
PE/Atto594	NB100-122PEATT594
PE/Cy5.5	NB100-122PECY55
PE/Cy7	NB100-122PECY7
PerCP	NB100-122PCP
mFluor Violet 450 SE	NB100-122MFV450
mFluor Violet 500 SE	NB100-122MFV500
mFluor Violet 610 SE	NB100-122MFV610

Available Formulations

Formulation	Catalog #	Availability	Price
BSA Free	NB100-122SS

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NB600-501

beta-Actin Antibody (AC-15)

(27 Reviews)

(595 Publications)

0.1 ml

NB600-501

Western Blot: beta-Actin Antibody (AC-15) [NB600-501] - Cell line lysates were separated on SDS-PAGE and probed with 1:5000 Monoclonal Anti-beta-Actin Clone: AC-15 . The antibody was developed using Goat Anti-Mouse IgG-Peroxidase and a chemiluminescent substrate.Lanes: 1. HeLa, 2. JURKAT, 3. COS7, 4. NIH-3T3, 5. PC-12, 6. RAT2, 7.CHO, 8. MDBK, 9. MDCK

Immunocytochemistry/Immunofluorescence: beta-Actin Antibody (AC-15) [NB600-501] - Analysis of beta-Actin antibody on mouse fibroblasts. Image from verified customer review.

Immunohistochemistry: beta-Actin Antibody (AC-15) [NB600-501] - Staining of formalin-fixed, paraffin-embedded human heart sections with 1:1000 Monoclonal Anti-beta-Actin Clone: AC-15.

Western Blot: beta-Actin Antibody (AC-15) [NB600-501] - Analysis of beta-Actin in bovine whole skeletal muscle lysate. Image from verified customer review.

Western Blot: beta-Actin Antibody (AC-15) [NB600-501] - Whole cell extract of human fibroblasts was separated on SDS-PAGE and blotted with Monoclonal Anti-beta-Actin. The antibody was developed with Goat Anti-Mouse IgG, Peroxidase conjugate and AEC substrate. Lanes A: Antibody dilution 1:5,000 B: Negative control (only secondary antibody).

Western Blot: beta-Actin Antibody (AC-15) [NB600-501] - beta-Actin expression in human glioma cells using anti-beta-Actin antibody. Image from verified customer review.

Western Blot: beta-Actin Antibody (AC-15) [NB600-501] - HEK293T cells transfected with Niaph virus Matrix Protein, expression 24h later. This image was submitted via customer review.

<b>Genetic Strategies Validation. </b>Western Blot: beta-Actin Antibody (AC-15) [NB600-501] - MMP-11 expression in oral cancer cell lines and the MMP-11 knockdown in SCC9 cells reduce cell migration. Western blot analysis showing the expression of MMP-11 protein in five oral cancer cell lines. Beta-Actin is included as a loading control. Image collected and cropped by CiteAb from the following publication (//dx.plos.org/10.1371/journal.pone.0113129), licensed under a CC-BY license.

Western Blot: beta-Actin Antibody (AC-15) [NB600-501] - HSP90 and HSP70 interact with PABPN1. Interaction of PABPN1 with HSP90. Lysates from HEK293 cells transfected with Flag-HSP90 and GFP-tagged A17-PABPN1 were immunoprecipitated with an anti-Flag antibody. Immunoprecipitates (IP) were analyzed by Western blot (IB).Beta-Actin is included as a loading control. Image collected and cropped by CiteAb from the following publication (//dx.plos.org/10.1371/journal.pone.0138936), licensed under a CC-BY license.

Western Blot: beta-Actin Antibody (AC-15) [NB600-501] - GFAP and IL-6 are elevated in cultures of spinal cord compared to cortical astrocytes. ((G, H) Analysis of protein homogenates from brain or spinal cord astrocyte cultures also demonstrated higher levels of GFAP expression, but no differences in ALDH1L1. Beta-Actin is included as a loading control. Image collected and cropped by CiteAb from the following publication (//dx.plos.org/10.1371/journal.pone.0180697), licensed under a CC-BY license.

Immunocytochemistry/Immunofluorescence: beta-Actin Antibody (AC-15) [NB600-501] - Cultured FS-11 human fibroblast cell line stained using Monoclonal Anti-Beta-Actin, Clone No. AC-15 (NB600-501).

Immunocytochemistry/Immunofluorescence: beta-Actin Antibody (AC-15) [NB600-501] - HS-68 cells.

Simple Western: beta-Actin Antibody (AC-15) [NB600-501] - Analysis using the HRP conjugate of NB600-501. Electropherogram image(s) of corresponding Simple Western lane view. Beta-Actin antibody was used at 1:500 dilution on Hela, MCF-7, SH-SY5Y, & Jurkat lysate(s).

Simple Western: beta-Actin Antibody (AC-15) [NB600-501] - Analysis using the HRP conjugate of NB600-501. Image shows a specific band for Beta-Actin in 0.2 mg/mL of Hela, MCF-7, SH-SY5Y, & Jurkat lysate(s). This experiment was performed under reducing conditions using the 12-230 kDa separation system. * Non-specific interaction with the 230 kDa Simple Western standard may be seen with this antibody.

VPA reduces autophagy at P28. (A) Schematic of VPA administration. (B) Representative Western blot images for LC3B, p62, p-rpS6 S240/244, DARPP32, & actin. (B–G) Quantification of (B–D) p-rpS6 S240/244 relative to actin, (B,E,F) LC3B-ii relative to actin (B,G,H) p62 & (B,I,J) DARPP32 normalized to VPA untreated (veh, vehicle) mice at P10 & P28, respectively. Data analyzed with unpaired, two-tailed t-test; (C) p-rpS6 S240/244/actin at P10, ns; (D) p-rpS6 S240/244/actin at P28, t11 = 2.882, p = 0.0149 (E) LC3B-ii/actin at P10, ns; (F) LC3B-ii/actin at P28, t10 = 2.231, p = 0.047; (G) p62 at P10, ns; (H) p62 at P28, t10 = 3.752, p = 0.0038; (I) DARPP32 at P10, ns, & (J) DARPP32 at P10, ns, *p 0.05). ns = not significant. Image collected & cropped by CiteAb from the following publication (//www.oncotarget.com/lookup/doi/10.18632/oncotarget.21145), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: beta-Actin Antibody (AC-15) [NB600-501] - Effect of IFN-gamma pretreatment on carfilzomib sensitivity of H727 (a) & H23 (b) cells. IFN-gamma (150 U⋅ml−1) pretreatment for 24 h led to increased expression (left panel) & activity (middle panel) of proteasome immuno-subunits, & sensitized H727 cells to Cfz, while desensitizing H23 cells towards Cfz. The IC50 values displayed a statistically significant difference between IFN-gamma -pretreated cells & vehicle control (P value 0.1, n = 3). (c) Immunoblotting results showing no detectable expression of P-glycoprotein (P-gp) in H727 cells. DLD-1 cells with acquired Cfz resistance via P-gp upregulation (DLD-1/CfzR) were used as a positive control. (d) The co-treatment of reversin-121 (7.5 µM, P-gp inhibitor) did not affect the sensitivity of H727 cells to Cfz. The IC50 values did not show statistically significant difference between in the presence & absence of reversin-121 (Student’s t-test). (e) The levels of remaining Cfz in culture media were comparable between H727 & H23 cells (no statistically significant differences, t-tests the Holm-Sidak method to correct for multiple comparisons with alpha = 0.05). Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/30858500), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: beta-Actin Antibody (AC-15) [NB600-501] - (a) Immunoblots showing the expression of cP & iP catalytic subunits in H727 & H23 cells. Immunoblots for additional cell lines displaying differential expression of proteasome catalytic subunits are presented in Supplementary Fig. S2. (b) Heat map displaying differential proteasome activity profiles in H727 & H23 cell lines. Purified human 20S cP & iP were used as controls for individual subunits: 20S cP for beta 5 & beta 1 & 20S iP for beta 5i & beta 1i. The numbers represent hydrolysis rates of respective substrates (RFU/min, mean values derived from three technical replicates) & were converted to color format & clustered by using the program “R” (//www.R-project.org). (c) Remaining catalytic activities of individual proteasome subunits in H727 & H23 cells 4 h after treatment with 20 nM of carfilzomib (left panel) or 20 nM bortezomib (right panel). Data are presented as mean ± SD derived from three technical replicates. Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/30858500), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: beta-Actin Antibody (AC-15) [NB600-501] - Effects of insulin on AKT & ERK phosphorylation & cell viability in primary human pancreatic duct cells. Phosphorylated AKT & ERK were measured in primary pancreatic exocrine cultures treated with the indicated concentrations of insulin for 5 minutes (A, B) & 24 hours (C, D) (n =3-4) Fold refers to the fold change of sample relative to control at the same time point. (E) Quantification of automated cell-counting studies employing live-cell imaging of Hoechst-labeled cell cultures over 60 hours. (n =3). (F) Quantification of proliferation by BrdU staining of treated relative to untreated over 3 days (n =4). (G) Quantification of the average number of dying/dead treated cells, propidium iodide (PI) labeled, over 60 hours relative to non-treated cells. (n =3). (H) Human exocrine cells were exposed to 0, 0.2, 2, 20, 200 nM insulin for 3 days. Bright-field images are representative of 3 cultures. (I) Effects of inhibition of RAF1/ERK signalling on PI incorporation with 10 μM GW5074 or AKT signalling with 100 nM Akti1/2 on human primary pancreatic exocrine cell viability (n =3). SF denotes serum free. Repeated Measures ANOVA analyses with Bonferroni’s post-test were performed. *Represents statistical significance of p 2 in CD95-Ab vs. saline liver samples (n = 5 per group). (F) Venn diagram including the 751 genes upregulated in livers of mice injected with CD95-Ab & the H3K9Ac ChIP-seq data on eight-week-old mouse livers in ENCODE showing a high degree of overlap (Hypergeometric test; p value: 9.643501e−58). (G) Chart generated using custom annotation scripts shows that response to external stimulus including inflammatory & defense response processes, & cytokine-cytokine receptor interaction pathways were enriched among the 467 genes overlapping between the two groups according to KEGG pathways. Means ± SDs are shown; t test; *p  0.05, One‐way ANOVA). ZO‐1 level: P 0.05, One‐way ANOVA). ZO‐1 level: P 1 indicate brighter nuclear staining for NF-kappa B. (c) BV2 cells were incubated with 200 ng/mL LPS in the presence of ENERGI-F704 for 24 h. After treatments, cell lysates were used to determine the levels of NF-kappa B using Western blotting. Data are presented as the mean ± SEM of three independent experiments (one-way ANOVA; * & **, P < 0.05 & P < 0.01; specific comparison to LPS-treated control). Representative images of three independent experiments are shown. Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/25025067), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: beta-Actin Antibody (AC-15) [NB600-501] - Sortilin forms homodimers in the extracellular & intracellular domains with intermolecular disulfide bonds in HEK293 cells.A, schematic of FLAG-sortilin Full, ECD+TMD, & ICD+TMD. SP, signal peptide; PP, propeptide. B & C, protein expression of FLAG-sortilin Full, ECD+TMD, & ICD+TMD was validated in reducing (B) & non-reducing (C) Western blotting using anti-FLAG antibody. FLAG-sortilin Full & ECD+TMD form homodimers & multimers. Empty vector was used as a control. D, HEK293 cells transiently overexpressing FLAG-sortilin Full or ECD+TMD were treated with a cross-linker, BS3, & the cell lysates were used for reducing Western blotting with anti-FLAG antibody, showing dimerization of sortilin Full & ECD+TMD (n = 3). E, HEK293 cells stably overexpressing FLAG-sortilin ICD+TMD (FLAG-sortilin ICD+TMD HEK293 cells) were incubated with DMSO (Control), 20 μmol/liter MG-132 (MG) or 10 μmol/liter chloroquine (Chlo) for 7 h, & then reducing Western blotting was performed using anti-sortilin antibody. MG-132 increased the protein expression of FLAG-sortilin ICD+TMD, but chloroquine did not (n = 3). F, FLAG-sortilin ICD+TMD HEK293 cells were incubated with DMSO or MG-132 (2–20 μmol/liter) for 7 or 24 h. MG-132 increased FLAG-sortilin ICD+TMD in a time- & concentration-dependent manner (n = 3). G, following 16-h incubation of HEK293 cells (Control) or FLAG-sortilin ICD+TMD HEK293 cells (ICD+TMD) with MG-132 (5 μmol/liter) & immunoprecipitation with anti-FLAG antibody, non-reducing Western blotting showed dimerization of sortilin ICD+TMD using anti-sortilin antibody (n = 3). Monomers, homodimers, & multimers are abbreviated as MO, D, & MU, respectively. IB, immunoblotting. Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/29382723), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: beta-Actin Antibody (AC-15) [NB600-501] - RAF1/ERK signalling is preferentially required for PANC1 cell survival in the absence of exogenous insulin. (A) Effects of different small molecule inhibitors on propidium iodide (PI) incorporation (PI) in PANC1 cells were tracked & expressed as the fold change in the percent of PI & Hoechst co-positive cells over total Hoechst positive cells at that hour relative to t =0 hour. Kinetic data were analyzed relative to serum-free control by two-way ANOVA (n =3) Data points that have been shaded solid black represent statistical significance when compared to non-treated conditions at that time point. # Indicates statistical significance in cells treated with Akti1/2 when compared to control at that time point. (B) Average number of PI positive cells over time of each treated group in Figure 5A is shown as a histogram expressed in arbitrary units (AU). GW5074 exhibited statistical significance, where as other treatments did not yield significance. U0126 p = 0.38, GW5074 *p = 0.0005, Akti-1/2 p = 0.395, Wort. p = 0.292 (n = 3). (C) The effect of 24 hours treatment with inhibitors on cleaved caspase 3 protein levels in PANC1 cells. This is a representative immunoblot of three independent biological replicates (n =3). (D-E) PANC1 cells were serum starved & treated with either DMSO, 10 μM GW5074, 10 μM U0126, 200 nM Akti-1/2 & 1 mM wortmannin (wort.) for 24 hours & 120 hours (n =4-5). Cell viability of PANC1 cells was expressed as the fold change of the treated relative to control. (C-E) One-way ANOVA analysis with Bonferroni post-test was performed. *Represents statistical significance of p < 0.05 where treated groups are compared to control (-) in the post-hoc test. Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/25373319), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: beta-Actin Antibody (AC-15) [NB600-501] - Analysis of HCAR expression in D2 & D2G retina & ON. a, b Bar graphs showing Hcar1 (a), ***p = 0.0001, & Hcar2 (b) mRNA normalized to Hprt mRNA by qRT-PCR; n = 8 ON per group. c Immunohistochemical analysis of HCAR1 (red) expression in the untreated (Unt) & keto D2 retina & ON. GFAP (green) labels Müller glia & astrocytes, Iba1 (cyan) labels microglia, & DAPI (blue) stains nuclei. Arrows indicate colocalization of HCAR1 with Iba1+ microglia & GFAP+ astrocytes. d, e Percentage of mean fluorescence of HCAR1 in the inner retina (d), **p = 0.0036, & in the ROI of the proximal ON (e), ***p = 0.0001; five retinae per group, with five sections/retina. f Western blot analysis of HCAR1 protein level in retina. g Quantification by densitometry of HCAR1 protein normalized to beta -actin, ***p = 0.0008; n = 3 blots per group, each with independent samples. h HCAR1 protein in the ON normalized to beta -actin levels, as determined by capillary electrophoresis, **p = 0.0025; n = 3 ON per group. All bar graphs are presented as the mean ± SEM, n = 5, analyzed by two-tailed unpaired t test. Scale bar, 20 μm Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/30424795), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Simple Western: beta-Actin Antibody (AC-15) [NB600-501] - iPSC-RPE from an ARB donor show reduced levels of Best1 expression compared to five different iPSC-RPE control lines. (A) To compare Best1 protein expression levels between different iPSC-RPE lines, beta -actin was used as a loading control. (B) While iPSCs displayed no detectable Best1 protein expression, all shown iPSC-RPE lines – regardless of donor origin – displayed detectable Best1 protein expression. (C) The three control iPSC-RPE lines derived from unrelated, unaffected donors all exhibited similar levels of Best1 protein expression. The dad exhibited reduced levels compared to the unrelated controls & the mom showed levels that were about 50% that of the dad. This was expected as the antibody used cannot detect the mutant Best1I366fsX18 which both the mom & the ARB patient are heterozygous for. Best1 expression levels in the ARB iPSC-RPE line were about a quarter of the levels observed in the Mom 214 iPSC-RPE clone. (D) Widefield immunofluorescent staining of Best1 reveals basolateral staining in each of the iPSC-RPE lines, indicating that Best1 protein is properly localized regardless if Best1R141H and/or Best1I366fsX18 are present. Immunofluorescent signal was notably weaker in the ARB iPSC-RPE clones than in the control clones. Image collected & cropped by CiteAb from the following publication (//www.nature.com/articles/s41598-018-21651-z), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: beta-Actin Antibody (AC-15) [NB600-501] - Nifedipine & the expression p-AMPK, SREBP-1/2(nuclear), PPAR-alpha (nuclear) & lipin-1 (nuclear) in 48 h. (a) The AMPK/actin was upregulated to 107.1%, 112.6%, & 116.4%, significantly at doses of 7.5, 15, & 30 μM (p < 0.05), the ratio p-AMPK/AMPK was downregulated to 82.1%, 58.7%, & 49.9%, respectively. (b,c) SREBP-1 was activated in control. The SREBP-1/HDAC was upregulated to 109.9% & 132.0% by nifedipine at doses of 7.5 & 30 μM, respectively (p < 0.01); The SREBP-2 were elevated to 127.5%, 146.7% & 161.0% by nifedipine at doses of 7.5, 15 & 30 μM, respectively (p < 0.05 in 7.5 μM, p < 0.01 in 15 & 30 μM) (d) nifedipine downregulated PPAR-alpha . The PPAR alpha /Lamin B was suppressed from 80.2%, 85.6%, to 96.4% compared to control (100%) upon administration of nifedipine at doses 7.5, 15 & 30 μM, respectively. (p < 0.05 in 15 & 30 μM) (e) Lipin-1 was activated in control. The lipin-1/lamin B was upregulated after nifedipine treatment to 102.5%, 115.8% & 121.1%, respectively, by nifedipine at doses of 7.5, 15, & 30 μM, respectively (p < 0.05 in 15 & 30 μM). p-values ≤ 0.05 (marked as *) were considered statistically significant. In addition, p-values ≤ 0.01 are marked as **. Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/30934807), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: beta-Actin Antibody (AC-15) [NB600-501] - JEV NS5 interacts with mitochondrial trifunctional protein (MTP), the enzyme complex involved in LCFA beta -oxidation.(A) Western blot analysis of protein levels of the indicated proteins in A549 cells with JEV infection (MOI = 10) or NS5-Flag overexpression. (B) Western blot analysis of V5-tag, Flag-tag, & actin in HEK293T cells co-transfected with HADH alpha -V5-His plus the indicated plasmids expressing Flag-tagged JEV viral proteins for 24 h, then immunoprecipitated with anti-Flag affinity gel. WCL, whole-cell lysates. (C) Immunoprecipitation (IP) analysis with control IgG or anti-Flag affinity gel in A549, GFP-A549 & NS5-Flag-A549 cells. The protein bands identified as HADH alpha & HADH beta are indicated by arrows. (D) IP with anti-Flag affinity gel & Western blot analysis with the indicated antibodies in HEK293T cells co-transfected with vector control or NS5-Flag plus HADH alpha -V5-His or HADH beta -HA for 24 h. (E) IP analysis with nickel beads & Western blot analysis with the indicated antibodies in HEK293T cells adsorbed with JEV for 3 h, then transfected with vector control, HADH alpha -V5-His or HADH beta -V5-His for 24 h. Image collected & cropped by CiteAb from the following publication (//dx.plos.org/10.1371/journal.ppat.1004750), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: beta-Actin Antibody (AC-15) [NB600-501] - The effect of AM1241, SB203580, & PDTC on the thermal threshold & the expression of P2Y12 & P2Y13 receptors. Data are presented as TWL. All values represent mean ± standard deviation. a Comparison of thermal stimulation pain threshold in the ipsilateral hind paw of rats (n = 8). Spinal ADPbetaS injection reduced TWL from days 3 to 7 after ADPbetaS injection. *P < 0.05 compared w/ control group. Pretreated w/ AM1241 (100 PM, 20 min), SB203580 (50 μM), or PDTC (5 μg/10 μl) significantly inhibited the reduced w/drawal threshold days 3 to 7 in ADPbetaS-treated rats, respectively (compared w/ ADPbetaS-treated rats, AM1241: P < 0.05; SB203580: P < 0.05; PDTC: P < 0.05). b RT-PCR results show the expression of P2Y12 mRNA expression in the dorsal spinal cord of the rats in every groups (n = 8 per group). *P < 0.05 means comparison w/ the control group; +P < 0.05 means comparison w/ the ADPbetaS-treated rats. c RT-PCR results show the expression of P2Y13 mRNA expression in the dorsal spinal cord of the rats in every group (n = 8 per group). *P < 0.05 means comparison w/ the control group; +P < 0.05 means comparison w/ the ADPbetaS-treated rats. d Western blotting image of P2Y12R expression. The top panel the target band, P2Y12R, & the bottom one for the loading control beta-actin. e Western blotting quantitative analysis of the P2Y12R expression in the dorsal spinal cord of the rats. *P < 0.05 means comparison w/ the control; +P < 0.05 means comparison w/ the ADPbetaS-treated rats. f Western blotting quantitative analysis of the P2Y13R expression in the dorsal spinal cord of the rats. *P < 0.05 means comparison w/ the control; +P < 0.05 means comparison w/ the ADPbetaS-treated rats Image collected & cropped by CiteAb from the following publication (//jneuroinflammation.biomedcentral.com/articles/10.1186/s12974-017-0960-0), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: beta-Actin Antibody (AC-15) [NB600-501] - Intrathecal injection of SB203580 (50 μM) or PDTC (5 μg/10 ml) suppressed the increased expression of P2Y12 & P2Y13R in the dorsal spinal cord of CCI rats. a Comparison of thermal stimulation pain threshold in the ipsilateral hind paw of the rats (n = 8). Data are presented as TWL. CCI reduced TWL from days 1 to 7 after nerve injury. *P < 0.05 compared with control group. Pretreated with SB203580 (50 μM) or PDTC (5 μg/10 ml) significantly inhibited the reduced withdrawal threshold days 1 to 7 in CCI rats, respectively (compared with vehicle-treated CCI group, SB203580: +P < 0.05; PDTC: +P < 0.05). b RT-PCR results show the expression of P2Y12 mRNA expression in the rat dorsal spinal cord (n = 8 per group). *P < 0.05 means comparison with the control group; +P < 0.05 means comparison with the vehicle-treated CCI group. c RT-PCR results show the expression of P2Y13 mRNA expression in the rat dorsal spinal cord (n = 8 per group). *P < 0.05 means comparison with the control group; +P < 0.05 means comparison with the vehicle-treated CCI group. d Western blotting image of P2Y12R & P2Y13R expression. The top panel was the target band, & the bottom one was for the loading control beta-actin. e Western blotting quantitative analysis of the P2Y12R expression in the rat dorsal spinal cord. *P < 0.05 means comparison with the sham group; +P < 0.05 means comparison with the vehicle-treated CCI group. f Western blotting quantitative analysis of the P2Y13R expression in the rat dorsal spinal cord. *P < 0.05 means comparison with the sham group; +P < 0.05 means comparison with the vehicle-treated CCI group Image collected & cropped by CiteAb from the following publication (//jneuroinflammation.biomedcentral.com/articles/10.1186/s12974-017-0960-0), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: beta-Actin Antibody (AC-15) [NB600-501] - Effects of HSM on the expression of P2X4R & P2X7R & ROS production in human macrophages.Cells were pretreated with various concentrations (1 or 2%) of HSM extract for 20 h, followed by treatment with LPS (0.5 μg/ml) for 3 h & ATP (5 mM) for 1 h. (a) The mRNA expression levels of P2X4R & P2X7R were determined by RT-PCR, using beta -actin as the internal control. (b) P2X4R & P2X7R mRNAs were quantified using real-time PCR. beta -actin gene expression was used for normalization. The results are expressed as fold changes, considering one as the value of untreated cells. (c) Cell lysates were analyzed by Western blot analysis used specific anti-P2X4R & anti-P2X7R antibodies. (d) ROS production was measured with the total ROS detection kit, using a fluorescence microplate reader. Pyocyanin (200 μM), a ROS inducer, was used as a positive control for ROS formation. Data are presented as means ± SE of three experiments preformed in duplicate. #P < 0.01 versus untreated cells. versus HSM-untreated control (ethanol) cells. *P < 0.01 versus HSM-untreated control (ethanol) cells. †P < 0.05 versus HSM (1%) treated cells. Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/23459183), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: beta-Actin Antibody (AC-15) [NB600-501] - Zinc-finger motifs of ZAP are required for JEV RNA binding & its antiviral activity.(A) Schematic representation of human ZAP isoforms (ZAP-L, 902 a.a. & ZAP-S, 699 a.a.). The four tandem CCCH-type zinc-finger (ZF) motifs within the N-terminus of ZAP are shown as solid black boxes. Deletion of the four ZFs (a.a. 73–193) are indicated with a dashed line. (B) 293T/17 cells infected with JEV (MOI = 5) for 16 h were transfected with plasmids expressing EGFP, WT or ZF-deleted ZAP-L-V5 & ZAP-S-V5 for additional 24 h. The viral RNA bound with V5-tagged proteins was pulled down by anti-V5 agarose affinity gel & amplified by RT-PCR with JEV 3′-UTR specific primers (middle panel). RT-PCR of input viral RNA in JEV-infected cells (lower panel). Western blot analysis of the immunoprecipitated ZAP-L & ZAP-S (WT & del4ZFs) (upper panel). (C-E) The indicated cells were infected with JEV (MOI = 5) for 10 h. Cell lysates, total RNA, & culture supernatants were determined for the indicated proteins by western blot (C), viral RNA by RT-PCR (D), & viral titer by plaque assay (E). Representative data from three independent experiments shown as mean ± SD (n = 3) were analyzed by two-tailed Student’s t test. *** P≤0.001. Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/30016363), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: beta-Actin Antibody (AC-15) [NB600-501] - Effects of HSM on IL-1 beta gene expression & secretion in human macrophages.Cells were pre-treated with either 1 or 2% of HSM extract for 20 h, followed by treatment with LPS (0.5 μg/ml) for 3 h & with ATP (5 mM) for 1 h. (a) The mRNA expression levels of IL-1 beta were determined by RT-PCR analysis. (b) IL-1 beta mRNAs were quantified using real-time PCR. beta -actin gene expression was used for normalization. The results are expressed as fold changes, considering one as the value of untreated cells. (c) The amount of IL-1 beta in cell culture supernatants was detected by ELISA. (d) The presence of IL-1 beta in cell lysates & cell culture supernatants were analyzed by Western blot analysis. Data are presented as means ± SE of three experiments performed in duplicate. #P < 0.01 versus untreated cells. *P < 0.01 versus HSM-untreated (ethanol-treated) control cells. †P < 0.05 versus HSM (1%) treated cells. Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/23459183), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: beta-Actin Antibody (AC-15) [NB600-501] - Intrathecal injection of AM1241 (100pM) attenuated CCI-induced hyperalgesia & increased expression of p-p38MAPK & NF-kappaBp65 in the rat dorsal spinal cord (n = 8 per group). a Western blotting image of p-p38MAPK expression. The top panel was the target band, p-p38MAPK, & the bottom one was for the loading control beta-actin. b Western blotting quantitative analysis of the p-p38MAPK expression in the rat dorsal spinal cord. *P < 0.05 means comparison with the sham group; +P < 0.05 means comparison with the vehicle-treated CCI group. c Western blotting image of NF-kappaBp65 expression. The top panel was the target band, NF-kappaBp65, & the bottom one was for the loading control beta-actin. d Western blotting quantitative analysis of the NF-kappaBp65 expression in the dorsal spinal cord of the rats. *P < 0.05 means comparison with the sham group; +P < 0.05 means comparison with the vehicle-treated CCI group Image collected & cropped by CiteAb from the following publication (//jneuroinflammation.biomedcentral.com/articles/10.1186/s12974-017-0960-0), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: beta-Actin Antibody (AC-15) [NB600-501] - Intralipid enhances the cytotoxicity of Abraxane in tumors. (A) The box-and-whisker plot represents the amount of paclitaxel in tumors determined by LC/MS. The number above the box denotes the median amount of remaining paclitaxel in the tumor tissues. (B) Western blot analysis of the apoptosis signal, cleaved caspase-3, in the tumors at 24 & 48 hrs after Abraxane treatment. beta -actin served as a loading control. The blots of caspase-3 & beta -actin are cropped from different parts of the same gel. The intensity of each band of the cleaved caspase-3 was measured by ImageJ software (NIH) & normalized to its own signal of beta -actin. The values summarized in the box-and-whisker plot denote the relative intensities compared with those in the control group. (C,D) The immunohistochemistry of cleaved caspase-3 (C) & the TUNEL assay (D) are analyzed in 48 hrs tumor sections. Original: 200x magnification; Inserts; 400x magnification. Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/32071352), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: beta-Actin Antibody (AC-15) [NB600-501] - The effect of minocycline on the expression of P2Y12 & P2Y13 receptors. All values represent mean ± standard deviation. a RT-PCR results show the expression of P2Y12 mRNA expression in the rat dorsal spinal cord (n = 8 per group). *P < 0.05 means comparison with the sham group; +P < 0.05 means comparison with the CCI rats. b RT-PCR results show the expression of P2Y13 mRNA expression in the rat dorsal spinal cord (n = 8 per group). *P < 0.05 means comparison with the sham group; +P < 0.05 means comparison with the CCI rats. c Western blotting quantitative analysis of the P2Y12R expression in the rat dorsal spinal cord. *P < 0.05 means comparison with the control; +P < 0.05 means comparison with the ADPbetaS-treated rats. d Western blotting quantitative analysis of the P2Y13R expression in the rat dorsal spinal cord. *P < 0.05 means comparison with the control; +P < 0.05 means comparison with the ADPbetaS-treated rats. e Western blotting image of P2Y12R expression. The top panel was the target band, P2Y12R, & the bottom one was for the loading control beta-actin. f Western blotting image of P2Y13R expression. The top panel was the target band, P2Y13R, & the bottom one was for the loading control beta-actin Image collected & cropped by CiteAb from the following publication (//jneuroinflammation.biomedcentral.com/articles/10.1186/s12974-017-0960-0), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: beta-Actin Antibody (AC-15) [NB600-501] - Effects of HSM on inflammasome components & caspase-4 activation in human macrophages.Cells were pretreated with various concentrations (1 or 2%) of HSM extract for 20 h, followed by treatment with LPS (0.5 μg/ml) for 3 h & ATP (5 mM) for 1 h. (a) The mRNA expression levels of ASC, NLRP1, NLRP3 & caspase-4 were determined by RT-PCR, using beta -actin as the internal control. (b) ASC, NLRP1, NLRP3 & caspase-4 mRNAs were quantified using real-time PCR. beta -actin gene expression was used for normalization. The results are expressed as fold changes, considering one as the value of untreated cells. (c) Cell lysates were analyzed by Western blot analysis using specific anti-ASC, anti-NLRP1 & anti-NLRP3 antibodies. (d) Cell lysates were analyzed for protein levels of caspase-4 by Western blot analysis. beta -actin was used as an internal control. Data are presented as means ± SE of three experiments preformed in duplicate. #P < 0.01 versus untreated cells. versus HSM-untreated control (ethanol) cells. *P < 0.01 versus HSM-untreated control (ethanol) cells. Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/23459183), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: beta-Actin Antibody (AC-15) [NB600-501] - The cleaved MFN fragments were further degraded by host proteasome machinery.(A) Western blot analysis of A549+on/MFN1 (lane 1–6) & /MFN2 (lane 7–12) cells with Dox treatment (all lanes) & DENV infection (lane 3–6 & 9–12). The cells were infected with DENV serotype 2 (moi 5) for 24 h, then incubated in culture media containing Dox (1 μg/ml) with (lanes 5–6 & 11–12) or without MG132 (0.5 μM) for 16 h. (B) Western blot analysis of four different serotypes DENV-infected A549 cells incubated with MG132-containing medium. (C) Western blot analysis of endogenous MFN1 & 2 cleavage in A549 stable cells overexpressing WT or protease-dead mutant (S135A) of DENV NS2B3 with or without 18-h treatment with MG132 (0.5 μM). Filled arrow: full-length; open arrow: cleaved product; star: non-specific band. The longer exposure images for endogenous MFN1 & 2 signals are also shown in panels B & C. Image collected & cropped by CiteAb from the following publication (//dx.plos.org/10.1371/journal.ppat.1005350), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: beta-Actin Antibody (AC-15) [NB600-501] - ENERGI-F704 decreases LPS-induced NO, iNOS, COX-2, & PGE2 production in BV2 cells. BV2 cells were stimulated with 200 ng/mL LPS & subsequently treated with either ENERGI-F704 or AICAR for 24 h. (a) Levels of NO in the culture of BV2 cell were determined. (b) After treatments, cell lysates were used to determine the level of iNOS & COX-2 using Western blotting. (c) LPS-treated BV2 cells were treated with ENERGI-F704 in cotreatment with or without compound C for 24 h. Cell lysates were used to determine the level of iNOS & COX-2 using Western blotting. (d) LPS-induced BV2 cells were treated with ENERGI-F704 in cotreatment with or without compound C for 48 h, & the level of PGE2 in the culture medium was determined. Data are presented as the mean ± SEM of three independent experiments (two-way ANOVA; *, **, & ***, P < 0.05, P < 0.01, & P < 0.01; specific comparison to LPS-treated control). Representative images of three independent experiments are shown. Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/25025067), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Simple Western: beta-Actin Antibody (AC-15) [NB600-501] - iPSC-RPE from all donors express RPE marker proteins. (A) As a loading control, each iPSC-RPE line (ARB patient, the patient’s parents, & three unrelated, unaffected controls) as well as iPSCs (negative control) were blotted for beta -actin. (B) Unlike iPSCs, all iPSC-RPE lines showed robust expression of CRALBP. (C) Just like CRALBP, iPSC-RPE from the patient, the patient’s mom, the patient’s dad, & the three unrelated, unaffected controls all expressed RPE65. The negative control (iPSCs) had no detectable RPE65 protein expression. (D) Each of the iPSC-RPE lines but not iPSCs displayed strong expression of the RPE marker Mer-tk. (E) The relative expression levels for each of these three markers is summarized for iPSCs & all of the iPSC-RPE lines. Graphs show the summary of expression for each marker from three separate blotting experiments. Variability between each of the controls as well as between the different iPSC-RPE lines was observed for each of the RPE markers. Control 4 displayed the highest levels of CRALBP, Control 138 had the highest levels of RPE65, & Control 1 showed the highest levels of Mer-tk. Image collected & cropped by CiteAb from the following publication (//www.nature.com/articles/s41598-018-21651-z), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: beta-Actin Antibody (AC-15) [NB600-501] - Effects of MFN1 or MFN2 overexpression on DENV infection.(A) Western blot analysis of A549+on/HA-MFN1 cells induced with Dox (1 μg/ml) or not for 18 h, then infected with DENV serotype 2 for 24 or 48 h by the indicated moi. The relative ratios of band intensity were quantified by ImageJ. p.i. (h): hours post infection. (B) DENV plaque-forming assay of culture supernatants from A549+on/HA-MFN1 cells cultured with or without Dox (1 μg/ml) for 18 h, then infected with DENV (moi 0.1 or 10) as indicated. Data in panels B-D & F-G are mean ± SD (n = 3 per group) & were compared by two-tailed Student’s t test. (C) RT-qPCR analysis of IFN beta mRNA expression at indicated time point in DENV-infected (moi 10) A549+on/HA-MFN1 cells with or without an 18 h-Dox-pretreatment. (D) Analysis of antiviral activity against dSinF-Luc/2A virus in culture media from A549+on/HA-MFN1 cells with DENV infection (moi 5, 48 h) with or without 18-h Dox pretreatment. (E) Western blot analysis of A549+on/HA-MFN2 cells induced with Dox (1 μg/ml, 18 h) or not, then infected with DENV (moi 10) for the indicated time. (F & G) Analysis of the culture supernatants derived from panel E for DENV titer (F) & LDH release (G). (H) Flow cytometry of A549+on/HA-MFN2 cells double stained with JC-1 & annexin V. Cells were infected with DENV (moi 5) for 48 h with or without 18-h Dox pre-treatment. Decreased red/green fluorescence ratio of JC-1 represents disrupted MMP. Image collected & cropped by CiteAb from the following publication (//dx.plos.org/10.1371/journal.ppat.1005350), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Reactivity:

Hu, Mu, Rt, Po, Bv, Ca, Ch, Fe, Fi, Gp, Ha, Le, Ma, Pm, Rb, Sh, Sq, Tr, Ze, Dr(-)

Species Glossary

Reactivity: Hu, Mu, Rt, Po, Bv, Ca, Ch, Fe, Fi, Gp, Ha, Le, Ma, Pm, Rb, Sh, Sq, Tr, Ze, Dr(-)

Applications:

, , , , , , , , ,

Applications: , , , , , , , , ,

Host:

Mouse Monoclonal

Host: Mouse Monoclonal

Sizes:

Sample Size Available

Sizes: Sample Size Available

Conjugate:

Unconjugated

Available Conjugates:

Validated by:

Validated by:

Conjugate	Catalog #	Availability	Size	Price
Alexa Fluor 350	NB110-67828AF350
Alexa Fluor 405	NB110-67828AF405
Alexa Fluor 488	NB110-67828AF488
Alexa Fluor 532	NB110-67828AF532
Alexa Fluor 594	NB110-67828AF594
Alexa Fluor 647	NB110-67828AF647
Alexa Fluor 700	NB110-67828AF700
Alexa Fluor 750	NB110-67828AF750
Biotin	NB110-67828B
CoraFluor 1	NB110-67828CL1
DyLight 350	NB110-67828UV
DyLight 405	NB110-67828V
DyLight 488	NB110-67828G
DyLight 550	NB110-67828R
DyLight 594	NB110-67828DL594
DyLight 650	NB110-67828C
DyLight 680	NB110-67828FR
DyLight 755	NB110-67828IR
FITC	NB110-67828F
HRP	NB110-67828H
Janelia Fluor 549	NB110-67828JF549
Janelia Fluor 646	NB110-67828JF646
PerCP	NB110-67828PCP
mFluor Violet 450 SE	NB110-67828MFV450
mFluor Violet 500 SE	NB110-67828MFV500
mFluor Violet 610 SE	NB110-67828MFV610

Available Formulations

Formulation	Catalog #	Availability	Price
BSA Free	NB110-67828

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NB100-479

HIF-1 alpha Antibody - BSA Free

(28 Reviews)

(520 Publications)

0.1 ml

NB100-479

ChIP-qPCR assay of HIF-1 binding to hypoxia response element (HRE) of the PKM2 gene in human MDA-MB-231 breast cancer cells treated with vehicle or DMOG (HIF inducer) for 8 hours. Negative control: immunoprecipitation was performed with IgG. Image courtesy of Gregg Semenza, Johns Hopkins University School of Medicine.

<b>Biological Strategies Validation. </b>Normoxic pre-treatment sensitizes glioma cells to radiation after graded chronic hypoxic (GCH) exposure. The results of anchorage-independent colony forming assays are shown for U87, U87-luc, GL261 glioma cells and 0308 GSCs after 5 Gy radiation exposure under varying O2 conditions. For easier comparisons among cell types, raw values are expressed as a % of the cell type's negative (non-irradiated) control and the means & SEMs are plotted. Each result represents at least 3 independent samples, plated in triplicate. Holm-Sidak comparisons for multiple groups were used for statistical comparisons of raw values (*p N) localization in three microscopic viewing areas were counted as shown in the lower panel. The total counted cell numbers ranged from 35 to 64. The experiments were repeated more than twice, except for the cell localization count, & the results were reproduced. Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/28775317), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: HIF-1 alpha Antibody - BSA Free [NB100-479] - Hypoxia signaling & cytotoxicity to CdCl2. (a) Wild-type (WT) cells were left untreated (Ctrl) or exposed to 150 μM CoCl2 (Co2+), 1, 5 or 10 μM CdCl2 (Cd2+) or 150 μM CoCl2 & 10 μM CdCl2 for 24 hours. HIF1 alpha −/− cell extract treated with 150 μM CoCl2 was used as a negative control. Nuclear protein was extracted & separated by SDS-PAGE, transferred to nitrocellulose membrane & probed with a HIF1 alpha (upper panel) or beta -actin (lower panel) specific antibody. The bands observed in the cadmium only WT cells are nonspecific as they are also observed in the HIF1 alpha −/− cells. (b) BNip3 mRNA expression levels were analyzed by qRT-PCR in wild type (WT, white bars) & HIF1 alpha −/− cells (black bars). Cells were left untreated (0), or exposed to 5 μM CdCl2 (Cd2+) or 150 μM CoCl2 (Co2+) for 24 hours. Each value was normalized to the control level in the corresponding cell line. *P 0.05; *p 60 recipients/genotype). (C) When 500 WT & KO cells were implanted into the mammary fat pad, the median time until 50% of recipients developed tumors > 500 mm3 was 64 days for WT mice & 127 days for KO mice (n = 14 recipients/genotype, P < 0.001, logrank test). (D) Western blot for HIF-1 alpha in three independent tumors (500 to 750 mm3) per genotype. WT & KO cells that were cultured for 6 hours under hypoxic conditions served as positive & negative controls, respectively. CRM, cross-reactive material. MTEC, mammary tumor epithelial cell. (E) Mean fold change ± SEM in expression of HIF-1 targets in KO tumors as determined by qRT-PCR (n = 5 tumors/genotype). Decreased gene expression in KO tumors is presented as a negative fold-change relative to WT tumors. (F) An increase in Ki67+ cells in KO tumors is balanced by an increase in caspase 3-positive cells (n = 5 tumors/genotype, *P < 0.05, Student's t-test). Representative immunostaining images are shown in Additional file 2 Figure S4. Image collected & cropped by CiteAb from the following publication (//breast-cancer-research.biomedcentral.com/articles/10.1186/bcr3087), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: HIF-1 alpha Antibody - BSA Free [NB100-479] - Effect of HIF-1 alpha silencing on alpha -MSH-induced apoptosis in B16-F10 melanoma cells during hypoxia.a Cells were transfected with shLacZ or shHIF-1 alpha plasmids for 48 h before harvest. HIF-1 alpha shRNA reduced the basal HIF-1 alpha mRNA & protein expression levels. b, c Relative mRNA expression levels were analyzed by real-time PCR. Data are expressed as fold change compared with control (means ± SD of triplicate experiments). d Cell lysates were analyzed by immunoblot using the indicated antibodies. beta -Actin was used as an internal control for loading & transfer. e The population of apoptotic cells was analyzed by flow cytometry & qualified as mean ± SD from triplicate experiments. *p < 0.05, **p < 0.01. Knockdown of HIF-1 alpha diminished alpha -MSH-induced apoptosis in B16-F10 melanoma cells during hypoxia Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/30062060), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Immunohistochemistry: HIF-1 alpha Antibody - BSA Free [NB100-479] - Hif1 alpha expression in neonatal & adult testis.(A) Section of testis from 5-day old (P5) male new born pups showing Hif1 alpha expression in MVH+ gonocytes within the seminiferous tubules (top) & negative control images without primary antibodies (bottom). Scale bar: 50μm. (B) Western blot analysis of Hif1 alpha expression in P5 testes (left) compared to extract of the adult brain sub-ventricular zone (SVZ) (right). Loading control ( beta -Actin) is shown below. (C) Section of adult (3 month old) testis showing Hif1 alpha expression in spermatogonia. Scale bar: 30μm. (D) Western blot analysis of whole adult testis. HEK293 cells treated with DFX were used as a positive control & intestinal tissue was used as a negative control. Loading control ( beta -Actin) is shown below. Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/27148974), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: HIF-1 alpha Antibody - BSA Free [NB100-479] - Expression of hypoxia-inducible factor (HIF) proteins after bilateral renal ischemia or sham ischemia. Immunoblots for HIF-1 alpha (A–C) & HIF-2 alpha (D–F) of tissue extracts from the cortex & outer & inner medulla of the left kidneys of rats 24 h & 5 days following recovery from either sham ischemia (○) or bilateral renal ischemia (●); n = 6 per group. G: typical image of the gel following electrophoresis. H: typical image of the nitrocellulose membrane following transfer. Values are expressed as medians (25th percentile, 75th percentile). Paired comparisons were performed using the Mann-Whitney U-test. Because paired comparisons were made at two time points, P values were conservatively adjusted using the Dunn-Sidak method with k = 2. PTr, PT, & PTr*T are the outcomes of two-way analysis of variance on ranking with the factors treatment (Tr) & time (T). AU, arbitrary unit; I1, 24 h after ischemia; I5, 5 days after ischemia; S1, 24 h after sham ischemia; S5, 5 days after sham ischemia. Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/30110566), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: HIF-1 alpha Antibody - BSA Free [NB100-479] - Hypoxia reduces inflammatory signaling pathways & NLRP3 expression & induces autophagy in IECs. a HT-29 cells were subjected to normoxia & hypoxia at the indicated times in the absence or presence of 10 µg/ml LPS. Autophagy was measured by variations in the ratio of LC3-II/LC3-I & the total amount of LC3 (LC3-I plus LC3-II) relative to beta -actin. Results are representative of two independent experiments. b, c & d HT-29 cells were subjected to normoxia & hypoxia for the indicated periods in the absence or presence of 10 µg/ml LPS, followed by transcript analysis. Statistical analysis was performed using one-way ANOVA followed by Tukey’s post-test. Results represent mean + s.e.m. of two independent experiments done in triplicate, *P < 0.05; **P < 0.01; ***P < 0.001; ns not significant. e HT-29 cells were subjected to normoxia or hypoxia in the presence & absence of 10 µg/ml LPS & 20 µM of MG132. Results are representative of two independent experiments. f & g Putative binding sites for HIF-1 alpha & NF-kappa B were found in the p62 f & NLRP3 g promoters using Genomatix software tools. Numbers under the boxes indicate the distance from the transcription start site. HT-29 cells were subjected to normoxia (21% O2) or hypoxia (0.2% O2) for 6 h & 24 h. ChIP analysis was performed using antibodies against HIF-1 alpha & NF-kappa B for immunoprecipitation. PCR was performed using the promoter-specific primers for the p62 f & NLRP3 g promoter binding sites of HIF-1 alpha & NF-kappa B. Aliquots taken prior to immunoprecipitation were used as input control. PCR products were run on 2% agarose gel. The results are representative of three independent experiments. Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/28740109), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: HIF-1 alpha Antibody - BSA Free [NB100-479] - Transient hyperoxia does not sensitize normal human astrocytes to radiation.Results are shown for the colony forming assays for all previously assayed cell lines & a normal human astrocyte cell line (Astro). Cells were continuously maintained under normoxic conditions (NOx) or exposed to 25 min of hyperoxia (50% O2) & then returned to normoxic conditions for 25 min (HyperOx) before being treated with a 5 Gy dose of radiation. To allow for ease of comparisons among cell types, raw values are expressed as a percentage of the corresponding cells type’s negative (non-irradiated) control & the means & SEMs are plotted. Each result represents three independent samples, plated in triplicate (*p<0.05, Student’s t-test). Also shown are Western blots of nuclear HIF-1 alpha at the time of irradiation for each cell line. Corresponding Western blots of lamin A/C are shown as a loading control. Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/25350400), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: HIF-1 alpha Antibody - BSA Free [NB100-479] - YC-1 attenuates pro-inflammatory differentiation of macrophages. (a,b) qRT-PCR analyses, revealing mRNA expression of the pro-inflammatory M1-differentiation markers NOS2 (a) & IL-6 (b) in murine J774A.1 macrophages under normoxia (left bars) & hypoxia (right bars). LPS exposure was carried out to induce M1-polarization in presence of YC-1 or vehicle control (DMSO; **P ≤ 0.01, n = 9). (c,d) Western blots of nuclear extracts, revealing protein levels of HIF-1 alpha (c) & HIF-2 alpha (d) in J774.A1 macrophages treated with vehicle (DMSO) or YC-1 under normoxic (left) or hypoxic (right) culture conditions. Simultaneous LPS-treatment was performed to induce pro-inflammatory activation. Note LPS-induced stabilization of HIF-1 alpha - (c), but not HIF-2 alpha -levels (d), & reversal of LPS-induced HIF-1 alpha -stabilization by YC-1 (c). Histone H3 was used as a loading control. (e,f) qRT-PCR analyses, revealing mRNA expression of the pro-inflammatory M1-differentiation markers NOS2 (e) & IL-6 (f) in BMDMs under normoxia (left bars) & hypoxia (right bars). LPS exposure was carried out to induce M1-polarization in presence of YC-1 or vehicle control (DMSO; ***P ≤ 0.001, **P ≤ 0.01, n = 6). Image collected & cropped by CiteAb from the following publication (//www.nature.com/articles/s41598-017-13638-z), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Reactivity:

Hu, Mu, Rt, Po, Ca, Fi, Gt, Ha, Pm, Rb, Ze

Species Glossary

Reactivity: Hu, Mu, Rt, Po, Ca, Fi, Gt, Ha, Pm, Rb, Ze

Applications:

, , , , , , , , ,

Applications: , , , , , , , , ,

Host:

Rabbit Polyclonal

Host: Rabbit Polyclonal

Sizes:

Sample Size Available

Sizes: Sample Size Available

Conjugate:

Unconjugated

Available Conjugates:

Validated by:

Validated by:

Conjugate	Catalog #	Availability	Size	Price
Alexa Fluor 350	NB100-479AF350
Alexa Fluor 405	NB100-479AF405
Alexa Fluor 488	NB100-479AF488
Alexa Fluor 532	NB100-479AF532
Alexa Fluor 594	NB100-479AF594
Alexa Fluor 647	NB100-479AF647
Alexa Fluor 700	NB100-479AF700
Alexa Fluor 750	NB100-479AF750
Allophycocyanin	NB100-479APC
Allophycocyanin/Cy7	NB100-479APCCY7
Biotin	NB100-479B
CoraFluor 1	NB100-479CL1
DyLight 350	NB100-479UV
DyLight 405	NB100-479V
DyLight 488	NB100-479G
DyLight 550	NB100-479R
DyLight 594	NB100-479DL594
DyLight 650	NB100-479C
DyLight 680	NB100-479FR
DyLight 755	NB100-479IR
FITC	NB100-479F
HRP	NB100-479H
Janelia Fluor 525	NB100-479JF525
Janelia Fluor 549	NB100-479JF549
Janelia Fluor 646	NB100-479JF646
Janelia Fluor 669	NB100-479JF669
PE	NB100-479PE
PE/Atto594	NB100-479PEATT594
PE/Cy5.5	NB100-479PECY55
PE/Cy7	NB100-479PECY7
PerCP	NB100-479PCP
mFluor Violet 450 SE	NB100-479MFV450
mFluor Violet 500 SE	NB100-479MFV500
mFluor Violet 610 SE	NB100-479MFV610

Available Formulations

Formulation	Catalog #	Availability	Price
BSA Free	NB100-479SS

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NB500-201

Survivin Antibody - BSA Free

(22 Reviews)

(374 Publications)

0.1 ml

NB500-201

<b>Biological Strategies Validation, Genetic Strategies Validation and Orthogonal Strategies Validation.</b>Knockdown Validated: Survivin Antibody [NB500-201] - Western blot analysis using Survivin and flotillin antibodies was performed on lysates of DMSO- and PTX-treated MDAMB231 cells ectopically expressing either control siRNA or Survivin siRNA (panels labeled WCL), as well as on the exosomes these cells generated (panels labeled Exos). Image collected and cropped by CiteAb from the following publication (//www.mdpi.com/2072-6694/8/12/111) licensed under a CC-BY license.

Immunohistochemistry: Survivin Antibody [NB500-201] - Protein expression pattern of Survivin in HCC tissues and non-neoplastic liver parenchyma. IAP members immunoreactivity was estimated by tissue microarray in a subset of HCC patients (n = 40). A-D, Representative Survivin cytoplasmatic immunostaining in a tumor core (A), in a tumor proximal to cirrhosis (C, N: cirrhosis, K: HCC), and in adjacent and long-distance non-neoplastic parenchyma (B and D, respectively). Image collected and cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/19397802) licensed under a CC-BY license.

<b>Genetic Strategies Validation. </b>Knockdown Validated: Survivin Antibody [NB500-201] - Anti-tumor effects and functional evidence of sCA-survivin-siRNA in HCT116 and HT29 solid tumor models. Immunostaining of survivin in the tumor tissues on day 19 using [NB500-201]. Scale bar, 50 um. Image collected and cropped by CiteAb from the following publication (//dx.plos.org/10.1371/journal.pone.0116022) licensed under a CC-BY license.

Immunocytochemistry/Immunofluorescence: Survivin Antibody [NB500-201] - Analysis using the HRP conjugate of [NB500-201]. Staining of Telophase with accumulation of survivin in the midbodies of two daughter cells. Survivin detection using [NB500-201].

Western Blot: Survivin Antibody [NB500-201] - Analysis of 30ug of HeLa whole cell lysate [NB800-PC1] using rabbit polyclonal [NB500-201] at 1ug/ml. Detection was performed using ECL method with 1 minute exposure. Band detected at higher molecular weight than the predicted MW (16 kDa).

Immunohistochemistry-Paraffin: Survivin Antibody [NB500-201] - Immunohistochemical staining of Survivin in human rectal cancer using [NB500-201] and DAB with hematoxylin counterstain.

Flow Cytometry: Survivin Antibody [NB500-201] - An intracellular stain was performed on HeLa cells with [NB500-201] and a matched isotype control. Cells were fixed with 4% PFA and then permeablized with 0.1% saponin. Cells were incubated in an antibody dilution of 2.5 ug/mL for 30 minutes at room temperature, followed by Rabbit IgG (H+L) Cross-Adsorbed Secondary Antibody, Dylight 550.

Western Blot: Survivin Antibody [NB500-201] - Western blot analysis using Survivin Antibody [NB500-201]. Effects of siRNA transfection on the expression of Bcl-xL and survivin. Bcl-xL and survivin protein content detected by Western Blotting 48 h after transfection. Bcl-xL and survivin levels are shown normalized to the reference protein beta-actin and relative to the ns-si control. Image collected and cropped by CiteAb from the following publication (//www.mdpi.com/1422-0067/14/6/12297) licensed under a CC-BY license.

Western Blot: Survivin Antibody [NB500-201] - Analysis of Survivin in human hepatocytes from cancer patient (left) and HeLa cell lysate (right) using [NB500-201]. Image from verified customer review. Note: bands detected at higher molecular weight than predicted (16 kDa)

Western Blot: Survivin Antibody [NB500-201] - Western blot analysis using [NB500-201]. Survivin protein is degraded by ubiquitin proteasome in serum-free and serum-containing media. Western blots and protein quantitation graphs showing that addition of MG-132 proteasome inhibitor (10 uM) extended survivin protein half-life in serum-free and serum-containing media. One representative western blot out of triplicate experiments was shown for each treatment and condition. *Indicates the time at which survivin protein is half of the amount at 0 hours (half-life), P 100. ** p-value 100. ** p-value < .01 using student t-test. H. PFGE analysis of EBV episomes & linears at 7 days post-transduction with lentivirus shRNA for shControl or shSurvivin in MUTU I or RAJI cell lines. Human alpha satellite DNA is used for DNA loading control Western blot of Survivin & Actin are shown below for each cell at 7 days post-transduction. Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/28077791), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: Survivin Antibody - BSA Free [NB500-201] - The p53-p21 axis facilitates downregulation of survivin via cell cycle(A) HCT116 wild typeand p21-deficient (p21-/-) cells were treated with 5 μM L-OHP for 24 hours. Whole cell lysates were analyzed with antibodies against p53, p21, & survivin; vinculin serves as loading control. (B) Cell cycle distribution was analyzed after 24 hours treatment by flow cytometry analysis (n = 3). (C) To induce p21, RKO p21ind cells were treated with 3 nM Muristerone A for 24 hours & tested for the levels of p21 & survivin; vinculin, loading control. (D)BIRC5 mRNA levels were analyzed by quantitative real-time PCR after 24 hours treatment with MurA in RKO p21ind cells (n = 3). (E) Cell cycle distribution was measured by flow cytometry analyses of cellular DNA content (n = 3). (F) Scheme summarizing the supposed mechanisms of survivin regulation after L-OHP & CPT-11 treatment. Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/29963241), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: Survivin Antibody - BSA Free [NB500-201] - Apoptosis & survival signaling after L-OHP & CPT-11(A) Western blot analysis using antibodies against p53 & pro-apoptotic BAX & PIG-3 after treatment with 5 μM L-OHP or 10 μM CPT-11. (B) Immunodetection of NF-kappa B p65, RELB & anti-apoptotic survivin, XIAP, BCL-XL & MCL1; vinculin serves as loading control. (C) Effects of increasing doses L-OHP & CPT-11 on caspase-3 & PARP1 cleavage after 24 hours treatment; alpha -tubulin serves as loading control. (D) Cells were treated with a combination of L-OHP & the caspase-inhibitor Z-VAD-FMK (50 μM). Immunodetection of survivin, p53 & full-length caspase-3 was conducted. Detection of apoptosis was determined by cleavage products of caspase-3 & PARP1; beta -actin serves as loading control. Please note: Figure 4A & 4B, as well as Supplementary Figure 2A show signals acquired by different detection methods, but originate from the same Western blots. This is due to a switch in the immunoblot chemiluminescence detection system from X-ray films (darker background) to a CCD camera system (Fusion Solo S, Vilber Lourmat; lighter background). Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/29963241), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: Survivin Antibody - BSA Free [NB500-201] - Anti-tumor effects & functional evidence of sCA-survivin-siRNA in HCT116 & HT29 solid tumor models.(A) For western blot analysis, HCT116 cells were seeded into 6-well plates & transfected with 100 pmol/well of either control or survivin siRNA by Lp (Lipofectamine 2000) or sCA. Actin blots served as loading controls. (B) For proliferation assays, cells were uniformly seeded into 96-well plates (1 × 104 cells/well), & 5 pmol/well siRNA was used. Cell viability was examined at 48 & 72 h by WST-8 assay. Data represent mean ± SEM. *P = 0.0294, **P = 0.0304 (n = 4, Wilcoxon rank test). (C) In vivo tumor growth. Each vehicle, carrying 15 μg of control siRNA or survivin siRNA, was administered by intravenous injection to mice with HCT116 tumors. Data represent the mean ± SEM (n = 10 tumors, Wilcoxon rank test). (D) Immunostaining of survivin in the tumor tissues on day 19. Scale bar, 50 μm. (E) In vivo live imaging of sCA-siRNA (6-FAM labeled) in HT29 tumor by multiphoton microscopy at 90 min. (F) Fluorescent detection of naked-siRNA (6-FAM labeled) or sCA-siRNA (6-FAM labeled) in the HT29 tumor at 4 h. Green: 6-FAM labeled siRNA; Red: microvasculature; Blue: DAPI stained nuclei. Scale bar, 50 μm. (G) Mice were administered with 40 μg of naked-survivin-siRNA or sCA-survivin-siRNA on days 0, 1, & 2. Tumors were removed on day 3, & western blot analysis for survivin was performed. (H) Many tumor cells treated with sCA-survivin-siRNA (6-FAM labeled) had condensed nuclei & were positive for TUNEL assay. Scale bar, 50 μm. Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/25738937), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: Survivin Antibody - BSA Free [NB500-201] - Survivin is essential for the salinomycin-induced apoptosis in UM cells. a UM cells were treated with salinomycin for 24 h & the protein levels of apoptosis-related proteins were detected by Western blot. b & c UM cells were transduced with lentiviral pTSB-Survivin cDNA (b), Survivin-shRNA constructs (c), or their corresponding empty vectors, & then incubated in the presence of puromycin (1 μg/mL) for 5 days to reach stable clones. Such survivin-manipulated cells were then exposed to salinomycin for 24 h, & subjected to trypan blue exclusion assay (left) & Western blot assay (right). Data represent mean ± SD. ns, not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001, Student’s t test. d qRT-PCR analysis of BIRC5 mRNA level was done in the 92.1 & Mel270 cells treated with salinomycin for 24 h. **, P < 0.01; ***, P < 0.001, one-way ANOVA, post hoc comparisons, Tukey’s test Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/31718679), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Immunocytochemistry/ Immunofluorescence: Survivin Antibody - BSA Free [NB500-201] - Anti-tumor effects & functional evidence of sCA-survivin-siRNA in HCT116 & HT29 solid tumor models.(A) For western blot analysis, HCT116 cells were seeded into 6-well plates & transfected with 100 pmol/well of either control or survivin siRNA by Lp (Lipofectamine 2000) or sCA. Actin blots served as loading controls. (B) For proliferation assays, cells were uniformly seeded into 96-well plates (1 × 104 cells/well), & 5 pmol/well siRNA was used. Cell viability was examined at 48 & 72 h by WST-8 assay. Data represent mean ± SEM. *P = 0.0294, **P = 0.0304 (n = 4, Wilcoxon rank test). (C) In vivo tumor growth. Each vehicle, carrying 15 μg of control siRNA or survivin siRNA, was administered by intravenous injection to mice with HCT116 tumors. Data represent the mean ± SEM (n = 10 tumors, Wilcoxon rank test). (D) Immunostaining of survivin in the tumor tissues on day 19. Scale bar, 50 μm. (E) In vivo live imaging of sCA-siRNA (6-FAM labeled) in HT29 tumor by multiphoton microscopy at 90 min. (F) Fluorescent detection of naked-siRNA (6-FAM labeled) or sCA-siRNA (6-FAM labeled) in the HT29 tumor at 4 h. Green: 6-FAM labeled siRNA; Red: microvasculature; Blue: DAPI stained nuclei. Scale bar, 50 μm. (G) Mice were administered with 40 μg of naked-survivin-siRNA or sCA-survivin-siRNA on days 0, 1, & 2. Tumors were removed on day 3, & western blot analysis for survivin was performed. (H) Many tumor cells treated with sCA-survivin-siRNA (6-FAM labeled) had condensed nuclei & were positive for TUNEL assay. Scale bar, 50 μm. Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/25738937), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Reactivity:

Hu, Mu, Rt, Ca, Fe, Gp, Ha

Species Glossary

Reactivity: Hu, Mu, Rt, Ca, Fe, Gp, Ha

Applications:

, , , , , , , , ,

Applications: , , , , , , , , ,

Host:

Rabbit Polyclonal

Host: Rabbit Polyclonal

Sizes:

Sample Size Available

Sizes: Sample Size Available

Conjugate:

Unconjugated

Available Conjugates:

Validated by:

Validated by:

Conjugate	Catalog #	Availability	Size	Price
Alexa Fluor 350	NB500-201AF350
Alexa Fluor 405	NB500-201AF405
Alexa Fluor 488	NB500-201AF488
Alexa Fluor 532	NB500-201AF532
Alexa Fluor 594	NB500-201AF594
Alexa Fluor 647	NB500-201AF647
Alexa Fluor 700	NB500-201AF700
Alexa Fluor 750	NB500-201AF750
Allophycocyanin	NB500-201APC
Biotin	NB500-201B-0.1ml
DyLight 350	NB500-201UV
DyLight 405	NB500-201V-0.1ml
DyLight 488	NB500-201G-0.1ml
DyLight 550	NB500-201R-0.1ml
DyLight 594	NB500-201DL594
DyLight 650	NB500-201C-0.1ml
DyLight 680	NB500-201FR
DyLight 755	NB500-201IR
FITC	NB500-201F-0.1ml
HRP	NB500-201H-0.1ml
Janelia Fluor 549	NB500-201JF549
Janelia Fluor 646	NB500-201JF646
PE	NB500-201PE
PerCP	NB500-201PCP
mFluor Violet 450 SE	NB500-201MFV450
mFluor Violet 500 SE	NB500-201MFV500
mFluor Violet 610 SE	NB500-201MFV610

Available Formulations

Formulation	Catalog #	Availability	Price
BSA Free	NB500-201SS

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NB100-2323

IRE1 alpha [p Ser724] Antibody - BSA Free

(9 Reviews)

(337 Publications)

0.1 ml

NB100-2323

P-Ire1a activation correlates with WFS1 cell expression. Cryosections of retina from 12 month old Wfs1+/+ and Wfs1-/- mouse were immunostained with anti-P-Ire1a antibody (red). DAPI was used for staining of cell nuclei (blue). RPE, retinal pigment epithelium; ONL, outer nuclear layer; INL, inner nuclear layer, RGC, retinal ganglion cells. Scale bars=50 um. Image collected and cropped by CiteAb from the following publication (//doi.org/10.1371/journal.pone.0097222) licensed under a CC-BY license.

Hepatic ER stress markers with cachexia progression. ER stress markers Bip, IRE1a, ATF6p50 and CHOP were examined in the liver of non, pre and severely cachectic mice. (n = 6-8 per group, p < 0.05) Dotted line indicates levels of Non-cachectic mice. Non = Non-Cachectic Apc Min/+ Sev = severely cachectic Apc Min/+; * denotes significantly different from Non-cachectic Apc Min/+ Image collected and cropped by CiteAb from the following publication (//doi.org/10.1371/journal.pone.0119888) licensed under a CC-BY license.

IRF-1 plays a central role in ER stress-mediated modulation of VCAM-1 expression by TGRL. HAEC were conditioned for 4 hr with TNFa (0.3 ng/ml) E: CHOP knockdown decreased TNFa-induced VCAM-1 expression. n=4. **P<0.01 vs. siCtrl+TNFa. Shown are representative blots from 3 independent experiments with similar results. Image collected and cropped by CiteAb from the following publication (//doi.org/10.1371/journal.pone.0078322) licensed under a CC-BY license.

Analysis using HRP conjugate of NB100-2323. Detection of phosphorylated IRE-1 alpha using NB100-2323. Lane 1: COS-7 untransfected Lane 2: COS-7 expressing wild-type IRE1 alpha Lane 3: COS-7 expressing kinase-dead IRE1 alpha. Theoretical molecular weight: 110 kDa.

<b>Biological Strategies Validation. </b>Analysis of anti-IRE1 alpha (pSer724) using Lot AN and AO of NB100-2323. HeLa cells were treated (+) or untreated (-) with 10 mM DTT for 60 min to activate the UPR. Total protein was separated on a 7.5% gel by SDS-PAGE, transferred to PVDF membrane and blocked in 5% BSA in TBST. The membrane was probed with 2.0 ug/ml antibody in 5% BSA, and detected with an anti-rabbit HRP secondary antibody using chemiluminescence. Theoretical molecular weight: 110 kDa.

Regulation of the unfolded protein response by KLF15. Western analysis of UPR activity in WT versus KLF15-/- primary hepatocytes. Hepatocytes were isolated from standard chow-fed 4-month-old male WT and KLF15 -/- mice. Two individual experiments were performed in triplicate; each lane indicates a technical replicate. Image collected and cropped by CiteAb from the following publication (//doi.org/10.1371/journal.pone.0077851) licensed under a CC-BY license.

HeLa cell lysate, 20 ug. Antibody at 1:1000, 1% skim milk in TBST, overnight incubation at 4C. WB image submitted by a verified customer review.

<b>Biological Strategies Validation. </b>Detection in Min6 cells which were treated with different concentrations of glucose for 3 hours prior to lysates preparation.

IRE1 (pS724) was detected in immersion fixed paraffin-embedded sections of human spleen using Rabbit Anti-Human IRE1 (pS724) polyclonal Antibody (Catalog # NB100-2323) at 1:300 for 1 hour at room temperature followed by incubation with the Anti-Rabbit IgG VisUCyte™ HRP Polymer Antibody (Catalog # VC003). Tissue was stained using DAB (brown) and counterstained with hematoxylin (blue). Specific staining was localized to the perinuclear cytoplasm in splenocytes.

Hepatic ER Stress.Representative hepatic western blots of BiP, phosphorylated (Ser51), total EIF2 alpha and phosphorylated:total EIF2 alpha , phosphorylated (Ser724), total IRE1 alpha and phosphorylated:total IRE1 alpha , and CHOP (n = 9). Diets not sharing a common letter differ significantly from one another (P≤.05).

Morphine suppressed 6-OHDA-induced ER stress through activation of UPR. (A,B) Morphine induced UPR in SH-SY5Y cells. Protein levels of GRP78, p-IRE1 alpha , IRE1 alpha , p-PERK, PERK, ATF6, CHOP and Tubulin in SH-SY5Y cells were analyzed (A) and quantified (B) by western blot. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001 vs. control.

Effect of cancer on ER stress markers.Bip1, IRE-1, ATF-6 p50 and CHOP expression in the liver of non—cachectic ApcMin/+ mice (N = 6 per group), compared to healthy C57BL/6 mice. Dotted line on the western blot indicates two different sections of the same gel. Values are expressed as Mean ± SE. * denotes significantly different from the healthy C57BL/6 mice as analyzed by a pre—planned t—test. p < 0.05.

Western Blot: IRE1 alpha [p Ser724] Antibody - BSA Free [NB100-2323] - ABT-263 induces ER stress & activation of the unfolded protein response.a Western blot of ER stress molecules in uveal melanoma cells treated with ABT-263 5 μM for 24 h & 48 h. HSP90 is used as a loading control. Representative western blots are shown. b XBP1 splicing in uveal melanoma cells exposed to ABT-263 5 μM for 15 h. *P-value < 0.05, **P-value < 0.01. c Kinetic analysis of ER stress proteins in Mel270 cells treated with ABT-263 5 μM in the absence or presence of qVD-OPh 20 μM. HSP90 is used as a loading control. Representative western blots are shown. Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/32337074), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: IRE1 alpha [p Ser724] Antibody - BSA Free [NB100-2323] - Effect of cancer on ER stress markers.Bip1, IRE-1, ATF-6 p50 & CHOP expression in the liver of non—cachectic ApcMin/+ mice (N = 6 per group), compared to healthy C57BL/6 mice. Dotted line on the western blot indicates two different sections of the same gel. Values are expressed as Mean ± SE. * denotes significantly different from the healthy C57BL/6 mice as analyzed by a pre—planned t—test. p < 0.05. Image collected & cropped by CiteAb from the following publication (//dx.plos.org/10.1371/journal.pone.0119888), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: IRE1 alpha [p Ser724] Antibody - BSA Free [NB100-2323] - ER stress is induced by acidosis activated OGR1-mediated signalling. Caco-2 cells subjected to different pH medium, following 4–6 h incubation in pH 7.6 serum free medium.(B) After 24 h pH shift, total protein was isolated & WB was performed. The results representative of 3 independent experiments. Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/31996710), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: IRE1 alpha [p Ser724] Antibody - BSA Free [NB100-2323] - Expression of ER stress markers in mouse & human skeletal muscle(a) TA muscle lysates from high fat fed mice were used to determine the expression of ER stress markers by Western blot analysis (n=6). (b-g) Western blot analysis was done to measure proteins involved in ER stress in vastus lateralis muscle from lean, obese, or diabetic subjects (n=6-13). Data are the means ± S.E.M. * indicates p<0.05 & ** indicates p<0.01 vs. chow or lean controls, using Student’s t-test (a) & One way ANOVA. Image collected & cropped by CiteAb from the following publication (//www.nature.com/articles/ncomms2851), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: IRE1 alpha [p Ser724] Antibody - BSA Free [NB100-2323] - The inositol-requiring enzyme 1 alpha (IRE1 alpha )/ X-box-binding-protein 1 spliced isoform (XBP1s) arm of unfolded protein response (UPR) is induced by angiotensin II in VSMCs. (A–C) The rat aortic VSMCs infected with adenovirus encoding GRP78 or control GFP (100 moi) for 48 h were stimulated with 100 nM AngII (AII) for 1–6 h & immunoblotting was performed as indicated. (A) Representative blots from 4 independent experiments. (B) Signal intensity was used to calculate the expression ratio of XBP1s to GAPDH. (C) Signal intensity was used to calculate the IRE1 alpha Ser724 phosphorylation ratio to the total IRE1 alpha . The bars in the graphs show the mean ± SD from 4 independent experiments. * indicates p < 0.05. Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/32679678), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: IRE1 alpha [p Ser724] Antibody - BSA Free [NB100-2323] - Morphine suppressed 6-OHDA-induced ER stress through activation of UPR. (A,B) Morphine induced UPR in SH-SY5Y cells. Protein levels of GRP78, p-IRE1 alpha , IRE1 alpha , p-PERK, PERK, ATF6, CHOP & Tubulin in SH-SY5Y cells were analyzed (A) & quantified (B) by western blot. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001 vs. control. (C,D) Protein levels of GRP78, CHOP & Actin in SH-SY5Y cells were analyzed (C) & quantified (D) by western blot in the indicated groups. ∗P < 0.05, ∗∗P < 0.01 vs. control & #P < 0.05, ##P < 0.01 vs. 6-OHDA-treated group. All data are expressed as mean ± SEM. Image collected & cropped by CiteAb from the following publication (//journal.frontiersin.org/article/10.3389/fnmol.2018.00120/full), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: IRE1 alpha [p Ser724] Antibody - BSA Free [NB100-2323] - Hepatic ER Stress.Representative hepatic western blots of BiP, phosphorylated (Ser51), total EIF2 alpha & phosphorylated:total EIF2 alpha , phosphorylated (Ser724), total IRE1 alpha & phosphorylated:total IRE1 alpha , & CHOP (n = 9). Diets not sharing a common letter differ significantly from one another (P≤.05). Image collected & cropped by CiteAb from the following publication (//dx.plos.org/10.1371/journal.pone.0167979), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: IRE1 alpha [p Ser724] Antibody - BSA Free [NB100-2323] - Effect of Insig-1 overexpression on nSREBP-1c & proteins related to ER stress pathway in INS-1 cells. INS-1 & INS-1-Insig-1 cells were stimulated with 11.2 mM & 25.0 mM glucose for 0, 24 & 72 h. A) nSREBP-1c & p-IRE1 alpha pathway of ER stress related proteins were detected by Western blot. GAPDH was used as control for protein loading. Equal amount of proteins (50 μg) were loaded. B) Relative protein expression compared to GAPDH, data are the means ± SE of three independent experiments; *, P < 0.05, **, P < 0.01 VS INS-1 cells at the same time point. Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/21843373), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: IRE1 alpha [p Ser724] Antibody - BSA Free [NB100-2323] - cCK-18 is formed following activation of the UPR. A. Immunoblots for phospho-IRE1 (p-IRE1), total IRE1 (IRE1), & cleaved cytokeratin 18 (cCK-18) showing time-dependent increase of cCK-18 in A549 cells exposed to 0.5 μM of thapsigargin (TG, +) for various lengths of time. Immunoblots showing an increase in total & phospho-IRE1 confirm thapsigargin activates the UPR in A549 cells compared to vehicle controls (−). B. Immunoblots for cCK-18 showing time-dependent appearance of cCK-18 in annexin V– or V+ A549 cells exposed to 0.5 μM of thapsigargin for various lengths of time. Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/23167970), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: IRE1 alpha [p Ser724] Antibody - BSA Free [NB100-2323] - Protein expression of downstream markers of ER stress in islets from lean, obese & high-fat-diet-fed fZDF rats. Protein lysates from islets isolated from three age-matched fZDF rats (obese), HF-fZDF (HFD) rats & their heterozygous lean littermates (lean) were separated by SDS-PAGE & analysed by western blotting. As controls, lysates from the islets isolated from Wistar rats or MIN6 cells treated with 1 μM thapsigargin for 2 h or sorbitol for 1 h were run alongside. Proteins were detected using antisera against (a) Calreticulin, HYOU1, GRP78, p-IRE1 alpha , p-eIF2 alpha & CHOP & as protein loading control rpS6 & (b) p-JNK & total JNK1/2 as loading control. Western blots were quantified using Image-J software. Image collected & cropped by CiteAb from the following publication (//www.nature.com/articles/nutd201335), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: IRE1 alpha [p Ser724] Antibody - BSA Free [NB100-2323] - CETP inhibition reduces endoplasmic reticulum stress. HAECs transfected with CETP or control scrambled siRNA. (A) Western blot analysis of PERK (n = 3, * p < 0.01) & qPCR analysis of ATF6 mRNA. (B) Phosphorylated & total IRE (n = 3, ns). (C) Representative microscopic images of HAECs stained with GADD153, n = 5, & (D) Grp78/BIP, & DAPI. Scale bar, 10 μm. Mean ± SEM, n = 5, * p < 0.05. HAECs, human aortic endothelial cells. CETP, cholesteryl ester transfer protein. NS, non-silenced. siCETP, silenced CETP. PERK, protein kinase RNA-like ER kinase. ATF6, Activating transcription factor 6. IRE, inhibitor resistant esterase. BIP, binding immunoglobulin protein. GADD153, growth arrest- & DNA damage-inducible gene 153. Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/33430172), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: IRE1 alpha [p Ser724] Antibody - BSA Free [NB100-2323] - Activation of UPR signaling pathways after UIRI. a & b The expression of p-PERK & t-PERK in kidneys of UIRI mice were evaluated with western blot analysis & quantified. c UIRI induces the activation of ATF6 as demonstrated by enhanced expression of cleaved ATF6 (cATF6). GAPDH was used as an internal control. d Fold-change expression of cATF6 in kidneys of UIRI mouse as compared with that of sham group mouse. e & f The expression of p-IRE1 & t-IRE1 in mice kidneys were evaluated with western blotting & quantified. Data are expressed as means ± SEM, n = 3 ~ 6 in each group. *P < 0.05, **P < 0.01, & ***P < 0.001, as compared with sham group Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/35765067), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: IRE1 alpha [p Ser724] Antibody - BSA Free [NB100-2323] - Apoptosis mediated by Arl6ip5 interference is regulated by Chop. (a) Immunoblotting was used to detect the protein level for p-IRE1 alpha , IRE1, Chop, Arl6ip5 & beta -actin in the UAMS-32 cell with NC-siRNA or Arl6ip5-siRNA treatment for 72 h. Representative results were shown from three independent experiments. (b–e) UAMS-32 cells first received NC-siRNA or Chop-siRNA for 24 h, then the medium was changed & further received NC-siRNA or Arl6ip5-siRNA for 72 h. Q-PCR was used to analyze the mRNA levels of Chop (b), GADD34 (c) & Trib3 (d). The apoptotic cells were stained with Annexin-V & the proportion were calculated (e). n=4. In all panels, error bars represent the mean±S.E.M. **P<0.01, *P<0.05 based on ANOVA Image collected & cropped by CiteAb from the following publication (//www.nature.com/articles/cddis2014427), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: IRE1 alpha [p Ser724] Antibody - BSA Free [NB100-2323] - Downregulation of gamma -taxilin & alpha NAC, & ER stress responses in mouse brain slice cultured under hypoxic conditions. (a) Immunohistochemistry shows downregulation of gamma -taxilin & alpha NAC expression in brain slices cultured for 48 h under hypoxic conditions. Scale bar, 20 μm. (b) Western blot shows downregulation of gamma -taxilin & alpha NAC, phosphorylation of PERK & IRE1 alpha & induction of CHOP in mouse brain slices cultured for 48 h under normoxic (−) or hypoxic conditions (+) Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/25880086), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: IRE1 alpha [p Ser724] Antibody - BSA Free [NB100-2323] - Differential ER stress response pathways initiated by gamma -taxilin or alpha NAC depletion. Proteins involved in UPR sensor (PERK, IRE1 alpha ), eIF2 alpha , JNK, Bcl-2, & Bcl-2-related protein (Puma, Bim, Bak, & Bax) signaling pathways were analyzed by Western blotting of HeLa S3 cells that were depleted of gamma -taxilin or alpha NAC by RNA intereference for 72 & 96 h. Upper panels, taxilin & NAC proteins; middle panels, UPR proteins; lower panels; apoptotic proteins; & bottom panel, beta -actin Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/25880086), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: IRE1 alpha [p Ser724] Antibody - BSA Free [NB100-2323] - The UPR is activated in type II AECs isolated from IPF lungs. Phosphorylated IRE-1 alpha (panel A) & spliced XBP-1 (panel B) were present in increased amounts in AECs from 3 different IPF lungs, but not in the 3 control subjects (* P < 0.05, **P = 0.0495 by Wilcoxon rank sum test). # indicates amplicon of unspliced XBP1 digested with Pst1. Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/23167970), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: IRE1 alpha [p Ser724] Antibody - BSA Free [NB100-2323] - ER stress is induced by OGR1 via IRE1 alpha /JNK signalling. (A) Caco-2 cells were subjected to different pH medium, with or without an OGR1 inhibitor (10 µM), following 4–6 h in pH 7.6 serum free medium. After 24 h pH shift, total protein was isolated & Western blot performed. Results are representative of two independent experiments. (B) Caco-2 cells were subjected to different pH medium After 24 h pH shift, total protein was isolated & Western blot performed. Results are representative of two independent experiments. (C) Caco-2 cells were subjected to different pH medium with or without a JNK inhibitor (10 µM) following 4–6 h in pH 7.6 serum free medium. After 24 h pH shift, total protein was isolated & Western blot performed. Results are representative of two independent experiments. (D) Caco-2 cells were starved & subjected to an acidic pH with or without a JNK inhibitor as described in (C). After 24 h pH shift, total RNA was isolated & mRNA expression was investigated by qPCR. Statistical analysis was performed using one-way ANOVA followed by Tukey’s post-test. Data are presented as means ± SE of three independent experiments (***p < 0.001). (E) Caco-2 cells were starved & subjected to different pH medium following 4–6 h in pH 7.6 serum free medium. After 24 h pH shift, total protein was isolated & co-IP using IRE1 alpha antibody & JNK antibody was performed, followed by immunoblotting. Results are representative of two independent experiments. pH conditions: High pH 7.5–7.8; Normal pH 7.2–7.4; Low pH 6.6–6.8. Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/31996710), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

$Western Blot: IRE1 alpha [p Ser724] Antibody - BSA Free [NB100-2323] - Downregulation of gamma -taxilin & alpha NAC & ER stress responses in hypoxic cells. (a) Phase-contrast micrographs of SK-N-SH cells cultures in hypoxic conditions for 0–48 h. Floating cells were evident by 37 h after the beginning of hypoxic treatment. (b) FACS (fluorescence-activated cell sorting) analysis of apoptosis in hypoxic SK-N-SH cells. Bars in the FACS profiles (left panel) indicate the fraction locations of annexin-positive cells. Bar graph indicates the relative numbers of annexin-positive cells (right panel). Bar graph data are shown as means±S.D. (n=3). *Significantly different from controls (0 h) (P<0.001, Tukey–Kramer test). (c) DAPI (4',6-diamidino-2-phenylindole) staining shows fragmented nuclei of apoptotic SK-N-SH cells after hypoxic treatment. Scale bar, 10 μm. (d) Downregulation of gamma -taxilin & alpha NAC & induction of ER stress responses in hypoxic SK-N-SH cells. Western blot analysis was performed 0 to 48 h after hypoxic treatment of cells. Upper panels, taxilin & NAC proteins; middle panels, UPR proteins; lower panels, apoptotic proteins; & bottom panels, cleaved caspase-9 & beta -actin proteins. (e) MG-132 treatment restore the gamma -taxilin & alpha NAC protein level in hypoxic (16 h) HeLa S3 cells. MG-132 (−), DMSO; MG-132 (+), 20 μM. (f) Caspase inhibition does not affect the hypoxia-induced downregulation of gamma -taxilin & alpha NAC in HeLa S3 cells. (g) Colocalization of gamma -taxilin & alpha NAC in normoxic (0 h) & hypoxic (8 h) HeLa S3 cells. Note that the colocalization fraction increases with the appearance of apoptotic phenotypes. Scale bars, 10 μm Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/25880086), licensed under a CC-BY license. Not internally tested by Novus Biologicals.$

$Western Blot: IRE1 alpha [p Ser724] Antibody - BSA Free [NB100-2323] - gamma -Taxilin ablation causes ER stress responses & apoptosis in normoxic cells. (a) Knockdown of gamma -taxilin expression by small interfering RNA (siRNA) in HeLa S3 cells. Two different siRNA constructs are equally effective. HeLa S3 cells were treated with solvent alone (Mock), control siRNA (Co), or gamma -taxilin siRNA ( gamma -tax-1 or -2) for 96 h. (b) Induction of apoptosis by gamma -taxilin ablation in HeLa S3 cells. gamma -Taxilin ablation causes apoptosis in HeLa S3 cells, but Mock or Co treatment did not. Bar graph shows fractions of annexin-positive cells (means±S.D., n=3). *Significantly different from Mock- or Co treatment (P <0.001, Tukey–Kramer test). (c) Confocal microscopy demonstrates coincidence of gamma -taxilin depletion & apoptotic nuclei in HeLa S3 cells treated with gamma -taxilin siRNA. Scale bar, 10 μm. (d) gamma -Taxilin depletion triggers ER stress responses in HeLa S3 cells. Upper panels, taxilin & NAC proteins; middle panels, UPR proteins; & lower panel, beta -actin. (e) gamma -Taxilin ablation induces accumulation of ubiquitinated proteins in HeLa S3 cells. Cell lysates were analyzed on 7.5% SDS-PAGE, followed by immunoblotting with antibodies specific for anti-mono- & polyubiquitinated conjugates (upper panel) & beta -actin (lower panel) Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/25880086), licensed under a CC-BY license. Not internally tested by Novus Biologicals.$

$Western Blot: IRE1 alpha [p Ser724] Antibody - BSA Free [NB100-2323] - Localization of the WT & mutant Wrn proteins in different subcellular fractions.(A) Example of western blots showing the presence of the Wrn WT protein in the nuclear fraction & the presence of the Wrn delta hel mutant protein mainly in the cytosolic fraction. Fractionations were performed on WT, Wrn delta hel/ delta hel, & Wrn-/- MEFs. Topoisomerase I & beta -tubulin are nuclear & cytoplasmic markers, respectively. As expected the molecular weight of the Wrn delta hel mutant protein was lower than the Wrn WT protein. No band was detected in Wrn-/- MEFs fractions with the anti-Wrn polyclonal antibody. (B) Example of western blots showing only the presence of the Wrn delta hel mutant protein in total ER & the peroxisomal fractions. Fractionations were performed on WT, Wrn delta hel/ delta hel, & Wrn-/- liver tissues. Catalase & calreticulin are used as peroxisomal & ER specific markers. This experiment was repeated three times. (C) Protein levels of GRP78, phosphorylated IRE alpha , & beta -actin (top panels) & of total IRE alpha , & beta -actin (bottom panels) in the spleen of three animals of each genotype. (D) Ratio of GRP78 signal over beta -actin signal from western blots. (E) Ratio of IRE alpha over beta -actin signal from western blots. (F) Ratio of phosphorylated IRE alpha signal over beta -actin signal from western blots. Experiments were performed in triplicates. Bars in all histograms represent SEM. Tukey post ANOVA test P-values are shown (*P < 0.05 & **P < 0.01). Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/26447695), licensed under a CC-BY license. Not internally tested by Novus Biologicals.$

Western Blot: IRE1 alpha [p Ser724] Antibody - BSA Free [NB100-2323] - IER3IP1 suppression decreases the UPR activation(A) mRNA levels of IRE1 alpha , Perk & Atf6 in IER3IP1 KD cells. 4 days after infection with IER3IP1 shRNA lentivirus, mRNA levels of IRE1 alpha , Perk & Atf6 in IER3IP1 KD cells were determined by qRT-PCR. *P<0.05, **P<0.01 compared to control group. Values are mean ± SEM. (B) Effect of IER3IP1 suppression on the IRE1 alpha arm of the UPR. 4 days after IER3IP1 KD in MIN6 cells, phosphorylation of IRE1 alpha , Bip, sXBP-1 & uXBP-were determined by Western blot. The loaded proteins in the two “- or +” lines were the same but from the different plates. (C) mRNA levels of downstream targets of sXBP-1 in IER3IP1 KD cells. 4 days after IER3IP1 KD in MIN6 cells, mRNA levels of downstream targets of sXBP-1, Erdj4, p58IPK, EDEM & Sec61a, were determined by qRT-PCR. Values are mean ± SEM. (D) Effect of IER3IP1 suppression on the Perk arm of the UPR. 4 days after IER3IP1 KD in MIN6 cells, phosphorylation of Perk & eIF2a, & ATF4 were determined by Western blot. The loaded proteins in the two “- or +” lines were the same but from the different plates. (E) IER3IP1 KD decreases UPR activation induced by ER stress in MIN6. 4 days after IER3IP1 KD, MIN6 cells were treated with 10 μg/ml tunicamycin (TC) or 1 μM thapsigargin (TG) for 6 hours. Then the expression levels of Chop, sXBP-1, uXBP-1 & IER3IP1 were determined by Western blot. Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/28915629), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: IRE1 alpha [p Ser724] Antibody - BSA Free [NB100-2323] - ATG5 & ATG7 influenced on autophagy, apoptosis & cell cycle through PERK/Nrf2 signaling. a The ER stress proteins were analysed by western blotting in Ad-GFP, Ad-ATG5 + Ad-ATG7,Ad-ATG5 + Ad-ATG7 + siPERK,Ad-ATG5 + Ad-ATG7 + siNrf2 & Ad-ATG5+ Ad-ATG7 + siPERK+siNrf2 induced chondrocytes. b The levels of ER stress proteins were normalized to beta -actin. c Determination of autophagy & apoptosis proteins expression by western blotting in Ad-GFP, Ad-ATG5 + Ad-ATG7, Ad-ATG5 + Ad-ATG7 + siPERK,Ad-ATG5 + Ad-ATG7 + siNrf2 & Ad-ATG5+ Ad-ATG7 + siPERK+ siNrf2 induced chondrocytes. dThe levels of related proteins were normalized to beta -actin. e FCM analysis was used to calculate the percentage of apoptotic cells at the time point of 24 h. The apoptosis rate were increased when combined treatment with silencing of PERK or Nrf2. Experiments were repeated 3 times, Representative images are shown. f Analysis of cell apoptosis. Data come from 3 independent experiments. g FCM analysis indicated that the S phase percentage were decreased compared to that of the control groups when combine infected with silencing of PERK or Nrf2 in C28I2 cells. Experiments were repeated 3 times, Representative images are shown. h Percentage of cells at each phase in different groups. *P < 0.05, **P < 0.01 compared with the controls. Values are means ± SD n = 3). (1:Ad-GFP, 2:Ad-ATG5 + Ad-ATG7, 3:Ad-ATG5 + Ad-ATG7 + siPERK, 4:Ad-ATG5 + Ad-ATG7 + siNrf2, 5:Ad-ATG5 + Ad-ATG7 + siPERK+ siNrf2) Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/31060556), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: IRE1 alpha [p Ser724] Antibody - BSA Free [NB100-2323] - Thapsigargin treatment induces calpain activation & autophagy. (A) Control & shCAPNS1 cells were treated with 100 nM thapsigargin for 10, 20 & 30 min & the lysates subjected to western blot analysis to quantify the precursor & active form of CAPN1. The ratio between active CAPN1 & tubulin is reported below each lane. (B) Time course measurement of intracellular calcium concentration. The plot indicates the ratio of mean Indo Violet/Indo Blue emission values of each cell population at individual time points (minutes). The ratio corresponds to the relative calcium concentration of control & shCAPNS1 U2OS cells before & after addition of thapsigargin (100 nm final). Moving average was used as smoothing method. (C) shCAPNS1 cells were treated with 100 nM thapsigargin for 30, 60 & 120 min & the lysates subjected to western blot analysis to detect LC3, p62, pIRE, CAPNS1 & tubulin. (D) Levels of p62 & LC3 normalized to tubulin levels. Image collected & cropped by CiteAb from the following publication (//journals.biologists.com/bio/article/doi/10.1242/bio.022806/256614/Calpain-mobilizes-Atg9-Bif-1-vesicles-from-Golgi), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: IRE1 alpha [p Ser724] Antibody - BSA Free [NB100-2323] - Abnormal Paneth cells show ER stress.(A, B) Representative transmission electron microscopy images of Paneth cells at the base of ileal crypts in (A) ICR & (B) SAMP1/YitFc mice. Scale bars indicate 2 μm. (C, D) Quantitative analysis of (C) granule number & (D) ER lumen diameter in Paneth cells (n = 3/each week for SAMP1/YitFc mice). For the measurements, three Paneth cells were randomly selected from each mouse. (E) SDS–PAGE Western blot analysis of ER stress markers, pIRE1 alpha , ATF4, cleaved-ATF6, & GRP78 in ileal crypts (n = 4/each group). Total-IRE1 alpha & HPRT1 was used as loading control. (F) Relative expression level of ER stress markers calculated from the band intensity. Error bars represent mean ± SEM. (C, D, F) Statistical significance was evaluated by t test in (C, D), & one-way ANOVA followed by Tukey’s post hoc test in (F). P < 0.05 was considered statistically significant. *P < 0.05, †P < 0.01, §P < 0.001. E, ER; G, granules; N, nucleus; n.s., not significant. Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/32345659), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: IRE1 alpha [p Ser724] Antibody - BSA Free [NB100-2323] - Hypoxia-induced downregulation of gamma -taxilin & alpha NAC & the subsequent ER responses are GSK-3 beta -dependent. (a) GSK-3 beta activation in hypoxia. Western blot analysis shows downregulation of phosphorylated (Ser 9) GSK-3 beta (phospho-GSK-3 beta ) in hypoxic (0–48 h) SK-N-SH & hypoxic (0–24 h) HeLa S3 cells. (b) Inhibition of GSK-3 beta activation by lithium chloride (LiCl, 100 mM) or CHIR-99021 (CHIR, 15 or 30 μM) maintained gamma -taxilin & alpha NAC protein levels in hypoxic SK-N-SH & HeLa S3 cells. (c) GSK-3 beta inhibition suppresses the expression of ER stress response proteins that are induced in hypoxic SK-N-SH cells. (d) GSK-3 beta RNA interference almost completely blocked the protein expression & restored the expression of gamma -taxilin & alpha NAC proteins in hypoxic HeLa S3 cells Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/25880086), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Reactivity:

Hu, Mu, Rt, Po, Dr, Gt, Ma, Pm, Pm, Rb, SyHa

Species Glossary

Reactivity: Hu, Mu, Rt, Po, Dr, Gt, Ma, Pm, Pm, Rb, SyHa

Applications:

, , , , , , , , ,

Applications: , , , , , , , , ,

Host:

Rabbit Polyclonal

Host: Rabbit Polyclonal

Sizes:

Sample Size Available

Sizes: Sample Size Available

Conjugate:

Unconjugated

Available Conjugates:

Validated by:

Validated by:

Conjugate	Catalog #	Availability	Size	Price
Alexa Fluor 350	NB100-2323AF350
Alexa Fluor 405	NB100-2323AF405
Alexa Fluor 488	NB100-2323AF488
Alexa Fluor 532	NB100-2323AF532
Alexa Fluor 594	NB100-2323AF594
Alexa Fluor 647	NB100-2323AF647
Alexa Fluor 700	NB100-2323AF700
Alexa Fluor 750	NB100-2323AF750
Allophycocyanin	NB100-2323APC
Biotin	NB100-2323B
CoraFluor 1	NB100-2323CL1
DyLight 350	NB100-2323UV
DyLight 405	NB100-2323V
DyLight 488	NB100-2323G
DyLight 550	NB100-2323R
DyLight 594	NB100-2323DL594
DyLight 650	NB100-2323C
DyLight 680	NB100-2323FR
DyLight 755	NB100-2323IR
FITC	NB100-2323F
HRP	NB100-2323H
Janelia Fluor 525	NB100-2323JF525
Janelia Fluor 549	NB100-2323JF549
Janelia Fluor 646	NB100-2323JF646
Janelia Fluor 669	NB100-2323JF669
PE	NB100-2323PE
PerCP	NB100-2323PCP
mFluor Violet 450 SE	NB100-2323MFV450
mFluor Violet 500 SE	NB100-2323MFV500
mFluor Violet 610 SE	NB100-2323MFV610

Available Formulations

Formulation	Catalog #	Availability	Price
BSA Free	NB100-2323SS

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NB100-449

HIF-1 alpha Antibody

(28 Reviews)

(302 Publications)

0.1 ml

NB100-449

Simple Western: HIF-1 alpha Antibody [NB100-449] - Simple Western lane view shows a specific band for HIF-1 alpha in 0.5 mg/ml of hypoxic HeLa lysate. This experiment was performed under reducing conditions using the 12-230 kDa separation system.

Immunohistochemistry: HIF-1 alpha Antibody [NB100-449] - Mouse Brain, Neurons 40X

Immunohistochemistry: HIF-1 alpha Antibody [NB100-449] - Detection of human HIF1-alpha by immunohistochemistry. Sample: FFPE section of renal cell carcinoma. Antibody: Affinity purified rabbit anti-HIF1-alpha antibody (NB110-58773). Detection: DAB

<b>Biological Strategies Validation. </b>Western Blot: HIF-1 alpha Antibody [NB100-449] - Detection of Human HIF-1 alpha by Western Blot and Immunoprecipitation. Samples: Whole cell lysate (5, 15 and 50 ug for WB; 1 mg for IP, 20% of IP loaded) from HeLa cells that were either treated with cobalt chloride (+; 200 uM) or mock treated (-). Antibodies: Affinity purified rabbit anti-HIF1 alpha antibody used for WB at 0.1 ug/mL(A) and 1 ug/mL (B) and used for IP at 3 ug/mg lysate. HIF-1 alpha was also immunoprecipitated by a previous lot of this antibody. Detection: Chemiluminescence with exposure times of 30 seconds (A) and 10 seconds (B).

Western Blot: HIF-1 alpha Antibody [NB100-449] - Blot showing the effect of hypoxia on the protein expression levels of the purine biosynthetic enzymes. HIF-1 alpha is stabilized in hypoxia as expected, and no significant increase in the purine enzymes was detected between normoxic (21% oxygen) and hypoxic (1% oxygen) growth conditions. The positions of molecular markers surrounding each band of interest are shown for each blot. ADSL (NBP2-03107), ATIC (NBP2-01941), FGAMS (NBP1-84691), GART (H00002618-M01), HIF-1a (NB100-449), PAICS (NBP2-02817), PPAT (NBP2-02056). Image collected and cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/32439803/) licensed under a CC-BY license.

Immunocytochemistry/Immunofluorescence: HIF-1 alpha Antibody [NB100-449] - Murine primary bone marrow derived macrophages stained with HIF1-alpha antibody (red). Nuclei were counterstained with DAPI (blue). Image from verified customer review.

Immunohistochemistry-Paraffin: HIF-1 alpha Antibody [NB100-449] - Analysis of microvascular density and HIF-1alpha activity. Low, homogenous expression of HIF-1alpha was detected in the tumors of NNK treated mice (40X, scale bar = 25 um). Image collected and cropped by CiteAb from the following publication (//molecular-cancer.biomedcentral.com/articles/10.1186/1476-4598-11-4), licensed under a CC-BY license.

Flow Cytometry: HIF-1 alpha Antibody [NB100-449] - HeLa cells were treated for 15 hrs with 200 uM CoCl2, fixed in PFA, and permeabilized in 90% MeOH. 1 X 10^6 cells were stained with 0.125 ug anti-HIF-1 alpha and secondary FITC-conjugated goat anti-rabbit (in a 150 uL reaction). Black: treated, anti-KLH control IgG; Red: untreated, anti-HIF-1 alpha; Blue: treated, anti-HIF-1 alpha.

<b>Biological Strategies Validation. </b>Western Blot: HIF-1 alpha Antibody [NB100-449] - Analysis of HIF-1 alpha in human myeloma cell lysate using anti-HIF-1 alpha. Cells were untreated or treated with IGF-1, IL-6 or CoCl2. Image from verified customer review.

Western Blot: HIF-1 alpha Antibody [NB100-449] - Detection of HIF-1 alpha in a hypoxic sample. Lane 1: CoCl2 treated Cos-7 nuclear extract (hypoxic). Lane 2: Untreated Cos-7 nuclear extract (normoxic).

Western Blot: HIF-1 alpha Antibody [NB100-449] - Detection of mouse HIF-1 alpha on hypoxia treated MEFs

<b>Biological Strategies Validation. </b>Western Blot: HIF-1 alpha Antibody [NB100-449] - Samples: Whole cell lysate (5, 15 and 50 ug) from HeLa cells that were treated with cobalt chloride (+; 200 uM) or mock treated (-). Antibodies: Affinity purified rabbit anti-HIF1 alpha antibody NB100-449 used for WB at 0.1 ug/ml. Detection: Chemiluminescence with exposure times of 30 seconds.

Western Blot: HIF-1 alpha Antibody [NB100-449] - BMDM were seeded at 0.5x10^6 overnight. Cells were treated with 10 ng/ml LPS for 24 hrs. Image from verified customer review.

Western Blot: HIF-1 alpha Antibody [NB100-449] - Analysis of total protein using anti-HIF-1 alpha, -CAIX, -ISCU antibodies and VDACs antibody (PMID: 29596470).

Western Blot: HIF-1 alpha Antibody [NB100-449] - Analysis of total protein using anti-HIF-1 alpha, -ISCU, -FXN antibodies. CoCl2 was used to treat or not HeLa cells for 2 days (PMID: 29596470).

Western Blot: HIF-1 alpha Antibody [NB100-449] - Detection of human HIF1 alpha by western blot. Samples: Whole cell lysate (15 and 50 ug) from HEK293T cells that were either treated with 200 uM cobalt chloride (+) or mock treated (-). Antibodies: Affinity purified rabbit anti-HIF1 alpha antibody NB100-449 used for WB at 0.1 ug/ml. Detection: Chemiluminescence with exposure times of 3 minutes.

Western Blot: HIF-1 alpha Antibody [NB100-449] - 3T3-L1 mouse embryonic fibroblast adipose-like cell line. Antibody at 1:2000. WB image submitted by a verified customer review.

<b>Biological Strategies Validation. </b>Immunocytochemistry/Immunofluorescence: HIF-1 alpha Antibody [NB100-449] - Formaldehyde-fixed asynchronous HeLa cells.

Immunoprecipitation: HIF-1 alpha Antibody [NB100-449] - HIF-1 alpha analysis in HEK293 cells. Image from verified customer review.

Immunoprecipitation: HIF-1 alpha Antibody [NB100-449] - Detection of human HIF1 alpha by western blot of immunoprecipitates. Samples: Whole cell lysate (1 mg for IP, 20% of IP loaded) from HEK293T cells that were treated with 200 uM cobalt chloride. Antibodies: Affinity purified rabbit anti-HIF1 alpha antibody NB100-449 used for WB at 1 ug/ml and for IP at 6 ug/mg lysate. Detection: Chemiluminescence with exposure times of 3 minutes.

beta -Glucan size affects the requirement for reactive oxygen species (ROS) in IL-1 beta induction.(B) Human mDCs from healthy donors or chronic granulomatous disease (CGD) patients were stimulated with curdlan or glucan-mp for 8 h. HIF-1 alpha and pro-IL-1 beta protein expression measured by immunoblot (n = 2 donors).

Western Blot: Rabbit Polyclonal HIF-1 alpha Antibody - Analysis of HIF-1 alpha antibody on mouse liver nuclear extracts. Image from a verified customer review.

HIF-1 alpha /2 alpha expression in myeloid-specific KO mice targeting the HIF pathway. (A) Images of the colon of wild-type (WT), myeloid-specific Hif-1a KO (hMRP8 Hif-1a KO) or von Hippel Lindau (Vhl) KO (hMRP8 Vhl KO) mice, immunostained for MRP8 (green) & the DNA-binding regions of Hif-1a mRNA (red). Mice were fed with 5% DSS for 4 days prior to immunostaining analyses. Note that there were no MRP8-positive cells that were positive for Hif-1a mRNA in hMRP8 Hif-1a KO (middle column) mice, but we observed many cells that were double positive for MRP8 & Hif-1a mRNA in hMRP8 Vhl KO mice (right column). (B) Images of the colon of hMRP8 Vhl KO mice fed with 5% DSS as in A, immunostained for MRP8 (green) & HIF-1 alpha (red, upper row) or HIF-2 alpha (red, bottom row). DAPI-stained nuclei are shown in blue. White boxes in A & B indicate the regions magnified in the lower or right images, respectively. Yellow arrowheads in A & B indicate cells positive for both markers. Scale bars: 100 μm. Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/29967068), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Immunohistochemistry: HIF-1 alpha Antibody [NB100-449] - Analysis of microvascular density & HIF-1 alpha activity. Microvascular density of pulmonary tumors was significantly higher in hyperplastic (B) & tumoral lesions (D) of NNK/NTHi treated mice compared to the NNK treated mice (A, & C) detected by CD105 immunostaining (10X, scale bar = 100 μm). HIF-1 alpha immunostaining after NNK/NTHi combined treatment showed hot-spots of high stromal expression in tumors (F), & high expression in perivascular-peribronchiolar lymphocytes (H). In contrast, low, homogenous expression of HIF-1 alpha was detected in the tumors (E) & perivascular-peribronchiolar lymphocytes (G) of NNK treated mice (40X, scale bar = 25 μm). Image collected & cropped by CiteAb from the following publication (//molecular-cancer.biomedcentral.com/articles/10.1186/1476-4598-11-4), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Immunocytochemistry/ Immunofluorescence: HIF-1 alpha Antibody [NB100-449] - Chronic ethanol feeding enhances tumorigenesis markers during AOM/DSS-induced tumorigenesis. AOM/DSS-induced colonic tumorigenesis was induced with (EF) or without (PF) 4 % ethanol feeding as described in Methods section. Cryosections of distal colon were stained for VEGF (a) pSmad (b), or HIF1 alpha (c), & co-stained for F-actin & nucleus. Images presented are representative of n of 5 for each group Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/26951793), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Immunohistochemistry-Paraffin: HIF-1 alpha Antibody [NB100-449] - Treatment with HBOT or PFD decreased HIF-1 alpha expression in the AD lesions.IHC for HIF-1 alpha (A). Scale bar is 100 µm. HIF-1 alpha + area was measured in five fields chosen at random by using image analysis software (B). RT-PCR for HIF-1 alpha (C). NT, not treated. Data are expressed as the mean ± SD (n = 12). *P<0.05, compared with the control group; ¶P<0.05, compared with the AD-NT group; §, P<0.05, compared with the AD-Steroid group. Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/25275529), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Immunohistochemistry-Paraffin: HIF-1 alpha Antibody [NB100-449] - Elevated PIK3/AKT/HIF-1 alpha pathways in KL SqCC tumours.(a,b) Analysis of TCGA gene expression (normalized TPM) & PIK3CA (a) & PTEN (b) genomic copy number alteration profiles. Each dot represents one SqCC patient (n=501). Boxes represent the interquartile range & whiskers are drawn to the minimum & maximum. Kruskal–Wallis non-parametric ANOVA, ****P<0.0001, ***P<0.001, **P<0.01. (c) IHC analysis (top) & quantification (bottom) of p63, p-AKT, p-S6, p-4EBP1, HIF-1 alpha & GLUT1 in KL tumours (n=6 each group). Two-tailed t-test, ****P<0.0001, ***P<0.001, **P<0.01. Scale bar, 50 μm. (d) Immunoblot analysis of HIF-1 alpha & GLUT1 in control shGFP & shHIF-1 alpha knockdown SqCC cell lines, HCC95 & HCC1588. All error bars represent the mean±s.e.m. Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/28548087), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Immunohistochemistry: HIF-1 alpha Antibody [NB100-449] - Immunolocalization of HIF1A in limbs.Immunoreactivity was detected in control limbs in the apical ectodermal ridge & interdigital regions (I.R.). The mesenchymal condensations/developing cartilaginous anlagen (Digits) were not immunoreactive. 4-OOHCPA treatment resulted in a concentration- & time-dependent increase in HIF1A immunoreactivity in this area in the 3 & 6 h exposure groups; staining was diminished by 24 h. Four separate replicates were done. Image collected & cropped by CiteAb from the following publication (//dx.plos.org/10.1371/journal.pone.0051937), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: HIF-1 alpha Antibody [NB100-449] - Effects of cell stress on VEGF-A & HIF1 alpha expression in Müller cells & Y79 photoreceptors. (A) VEGF-A was measured by ELISA using conditioned media collected from MIO-M1 & Y79 cells, n = 4–5/group in Müller cells & n = 8/group in Y79 photoreceptors, ** p < 0.01; (B) Western blots for HIF1 alpha using cellular proteins, n = 4/group, ** p < 0.01. For MIO-M1 Müller cells: Grey bars: 5 mM glucose. Black bars: 25 mM glucose. For Y79 photoreceptors: Grey bars: 11 mM glucose. Black bars: 25 mM glucose. NS, not significant; LG, low glucose; HG, high glucose. Image collected & cropped by CiteAb from the following publication (//www.mdpi.com/1422-0067/18/3/533), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: HIF-1 alpha Antibody [NB100-449] - Phenylhydrazine administration results in hypoxia in retinal vessels & in HIF-1 alpha & HIF-2 alpha stabilization in peripheral blood leukocytes.(A) 3-dimensional reconstructed imaging of the superficial vascular plexus of flat mounted retinae of mice (steady state, treated with PHZ & treated with PHZ & peak EIU induction) stained with DAPI, hypoxyprobe & Isolectin B4. (B) Representative analysis of HIFs by western blot: 50ug of protein from total cell lysate of blood leukocytes isolated from mice treated with PHZ compared to untreated; (C) relative densitometry quantification of HIF western blots from PBMC showing mean, n = 3, error bar showing SD, unpaired t test, ***P < 0.001. Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/28112274), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Immunohistochemistry-Frozen: HIF-1 alpha Antibody [NB100-449] - Ethanol elevates tumorigenic markers & nuclear localization of HIF1 alpha in colonic crypts during AOM/DSS-induced tumorigenesis: AOM/DSS-induced colonic tumorigenesis was induced with or without 4 % ethanol feeding as described in Methods section. a & b Colonic mucosal extracts were immunoblotted for pSmad & VEGF (a), & the band density evaluated using Image J software (b). Values are mean ± SE (n = 3). Asterisks indicate the values that are significantly different (p < 0.05) from corresponding values for AOM/DSS group. c Cryosections of colon were stained for HIF1 alpha (red) & nucleus (blue). Sections of the images from Fig. 2c are enlarged to show the detailed localization of HIF1 alpha in the nucleus. While arrows indicate nuclear co-localization of HIF1 alpha Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/26951793), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: HIF-1 alpha Antibody [NB100-449] - Dectin-1-induced NADPH oxidase-derived ROS is required for augmented IL-1 beta during TSLPR inhibition. (A) Human mDC derived from healthy donors (HD) or CGD patients were stimulated with SC glucan for 8 h (n = 2 representative donors presented, three separate experiments performed). Pro-IL-1 beta , IL-1 beta , HIF-1 alpha , phospho-AMPK, AMPK & beta -actin were measured by immunoblot. (B–E) Densitometry of cumulative data was performed using Image Studio Lite software with pro-IL-1 beta , IL-1 beta & HIF-1 alpha normalized to beta -actin & phospho-AMPK normalized to total AMPK. Data is reported as percentage of maximal signal observed within each donor (n = 3 independent donors, presented as pooled data). (F–H) Human mDC derived from HD or CGD patients were stimulated with SC glucan, CA glucan or heat killed C. albicans hyphae with anti-TSLPR or IgG isotype control antibodies for 24 h (n = 3 independent donors, presented as pooled data). IL-1 beta was measured in 24-h cell culture supernatants by ELISA. Cumulative data displayed as mean +SEM. Statistical analysis calculated using one-way ANOVA with Bonferroni post-tests (***p = 0.001,*p = 0.05). Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/31139177), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: HIF-1 alpha Antibody [NB100-449] - Reactive oxygen species production & expression of stabilized HIF1 alpha & AMPK subunit alpha 2 & in H1299 & H1299r cells(A) H1299, H1299r, P31 & P31r cell were seeded at a density of 250,000 cells/well & left for 24 h. The cells were then stained with 10 μmol/L dichloro-dihydro-fluorescein diacetate (DCFH-DA). Fluorescence was analysed for 10,000 events. Whole cell lysates were prepared from confluent cultures of H1299, H1299r cells. Proteins (90 μg) were resolved in 10% SDS-PAGE gels & transferred to a PVDF membrane. (B) Representative blot of HIF1 alpha protein expression. (C) Representative blot of AMPK subunit alpha 2 protein expression from four independent experiments. (D) Bar chart of AMPK subunit alpha 2 mean for four separate measurements. Statistical analyses was carried out using the student t-test. ** = p<0.01, * = p<0.05. Image collected & cropped by CiteAb from the following publication (//www.oncotarget.com/lookup/doi/10.18632/oncotarget.21885), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: HIF-1 alpha Antibody [NB100-449] - Pyruvate kinase isoform M2 (PKM2) & hypoxia-inducible factor 1 alpha (Hif-1 alpha ) bind directly to the promoter region of PDL1 in primary murine BMDM cells. BMDM cells were treated with TEPP-46 at 50 µM for 1 h prior to stimulation with LPS (100 ng/ml, 24 h) (A). Binding of PKM2 (left) or Hif-1 alpha (right) to HRE1 or HRE4 of the PD-L1 promoter was detected by incubating cell lysates with biotinylated oligonucleotides spanning the relevant HRE promoter region. Protein–oligonucleotide complexes were isolated using streptavidin agarose beads, & proteins were detected by western blotting. Representative of n = 3. Chromatin immunoprecipitation (ChIP)-PCR (B) using PKM2 & HIF-1 alpha antibodies & primers specific for three promoter regions of PD-L1 (HRE1, 2–3, & 4) & a known Hif-1 alpha binding region of vascular endothelial growth factor as a positive control showing binding of Hif-1 alpha (left) & PKM2 (right) to the PD-L1 promoter in LPS-treated BMDMs (100 ng/ml, 24 h). (C) Sequential ChIP assays measuring simultaneous endogenous binding of PKM2 & Hif-1 alpha to chromatin in response to LPS (100 ng/ml, 24 h) ±TEPP-46 (pretreatment using 50 µM, 60 min). ChIP data are calculated as percent of input, error bar represents mean ± SEM, & statistics are performed as two-tailed unpaired t-test *P < 0.05, **P < 0.01, & ***P < 0.001. Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/29081778), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: HIF-1 alpha Antibody [NB100-449] - Changes in Hepatic Oxygen Saturation & Hypoxia-related Protein Expression. (A) In the representative case, the hepatic oxygen saturation in the experimental group was lower than that in the control group at four, six, & eight weeks (51.6 vs. 54.5%, 48.4 vs. 53.9%, 32.3 vs. 43.2%, & 31.2 vs. 42.9%). (B) There was significant decrease of oxygen saturation of liver parenchyma in the experimental group compared to those of control group (45.2 vs. 51.9%, 43.0 vs. 49.0%, & 31.3 vs. 49.7%) at the four, six, & eight weeks, respectively (P < 0.001). (C) In the Western blot analysis, the experimental group was relatively higher expression of hypoxia-inducible factor-1 alpha (HIF-1 alpha ) & vascular endothelial growth factor (VEGF) protein than the control group at eight weeks. The full-length blots with these antibodies were presented in supplementary Figure S1. (D,E) The experimental group showed significantly higher expression of HIF-1 alpha & VEGF protein than the control group at eight-week (1.47 ± 0.48 vs. 0.19 ± 0.08 & 0.96 ± 0.23 vs. 0.33 ± 0.06, P = 0.019 & P < 0.018). *P < 0.05. Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/29079853), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: HIF-1 alpha Antibody [NB100-449] - BCAT1 expression & inhibition in human macrophages.(a) RNA-sequencing in human monocyte-derived macrophages (hMDMs) shows mRNA copies of BCAT enzymes in normalized counts. n=14 healthy donor hMDMs were used for RNA-seq. (b) Chemical structure of ERG240 & leucine. (c) Enzymatic activity of recombinant human BCAT1 & BCAT2 in presence of ERG240. (d) Relative expression of IRG1, HIF1A & IL1B measured by qRT-PCR in control, ERG240-treated (20 mM, 3 h), LPS treated (100 ng ml−1, 3 h), & LPS+ERG240 treated (LPS, 100 ng ml−1; ERG240, 20 mM for 3 h) hMDMs. ns, non-significant. All expression values are normalized to those obtained for HPRT gene expression. n=3 healthy donor hMDMs were used in each group. (e) Western blot analysis of IRG1, IL-1 beta , HIF-1 alpha & beta-actin (ACTB) in control (untreated), ERG240-treated (20 mM, 3 h), LPS treated (100 ng ml−1, 3 h) & LPS+ERG240 treated (LPS, 100 ng ml−1; ERG240, 20 mM for 3 h) hMDMs. The experiment is representative of three independent experiments using n=3 healthy donor hMDMs each. (f) GC/MS results showing itaconic acid production in hMDMs in presence or absence of ERG240 (20 mM). LPS treatment was for 8 h (100 ng ml−1). GC/MS plot for each donor is shown separately where itaconate abundance denotes arbitrary units. Error bars are s.e.m. Significance was tested using two-tailed Student’s t-test or one-way ANOVA. * P<10−3 following two-way ANOVA. Image collected & cropped by CiteAb from the following publication (//www.nature.com/articles/ncomms16040), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Immunocytochemistry/ Immunofluorescence: HIF-1 alpha Antibody [NB100-449] - HIF-1 alpha /2 alpha expression in myeloid-specific KO mice targeting the HIF pathway. (A) Images of the colon of wild-type (WT), myeloid-specific Hif-1a KO (hMRP8 Hif-1a KO) or von Hippel Lindau (Vhl) KO (hMRP8 Vhl KO) mice, immunostained for MRP8 (green) & the DNA-binding regions of Hif-1a mRNA (red). Mice were fed with 5% DSS for 4 days prior to immunostaining analyses. Note that there were no MRP8-positive cells that were positive for Hif-1a mRNA in hMRP8 Hif-1a KO (middle column) mice, but we observed many cells that were double positive for MRP8 & Hif-1a mRNA in hMRP8 Vhl KO mice (right column). (B) Images of the colon of hMRP8 Vhl KO mice fed with 5% DSS as in A, immunostained for MRP8 (green) & HIF-1 alpha (red, upper row) or HIF-2 alpha (red, bottom row). DAPI-stained nuclei are shown in blue. White boxes in A & B indicate the regions magnified in the lower or right images, respectively. Yellow arrowheads in A & B indicate cells positive for both markers. Scale bars: 100 μm. Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/29967068), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Immunocytochemistry/ Immunofluorescence: HIF-1 alpha Antibody [NB100-449] - Increased infiltration of myeloid cells expressing HIF-1 alpha in DSS-induced colitis. (A) Western blot analysis of MRP8, F4/80 or actin using the whole colon tissue lysate from mice fed with water or 5% DSS for 4 days. (B) Images of the colon of mice fed with water or 5% DSS, immunostained for myeloid cells using anti-MRP8 (green) or anti-HIF-1 alpha (red) antibodies. Nuclei were counterstained with DAPI. White rectangles indicate the areas magnified in the images shown below. Yellow arrowheads indicate cells with colocalization of MRP8 & HIF-1 alpha . Quantification of MRP8-positive cells & percentage of those expressing HIF-1 alpha are shown as bar graphs on the right-hand side. Data are mean±s.e.m. for at least three independent fields examined per mouse (n≥3 per group). **P<0.01 & ***P<0.001, assessed by Student's t-test. HPF, high-powered field. (C) Images of the colon of mice fed with 5% DSS, immunostained for MRP8 (red) & CD11b (green). DAPI-stained nuclei are shown in blue & a merged image is also shown. Scale bars: 100 μm. Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/29967068), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: HIF-1 alpha Antibody [NB100-449] - Increased HIF-1 alpha expression is regulated by TSLPR inhibition & not IL-1 beta secretion (A) Human mDC were stimulated with SC glucan with either anti-TSLPR or IgG isotype control antibodies for 8 h in the presence or absence of IL-1 beta neutralization antibodies (n = 1 representative donor presented, three separate experiments performed). Pro-IL-1 beta , IL-1 beta , HIF-1 alpha , phospho-p38 MAPK, p38 MAPK, phospho-AMPK, AMPK & beta -actin were measured by immunoblot. (B,C) Densitometry of cumulative data was performed using Image Studio Lite software with HIF-1 alpha normalized to beta -actin & phospho-AMPK normalized to AMPK. Data is reported as percentage of maximal signal observed within each donor (n = 3 independent donors, presented as pooled data). Cumulative data displayed as mean +SEM. Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/31139177), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: HIF-1 alpha Antibody [NB100-449] - miR-18b (miR-18b-5p) regulates Hif1 alpha & reduces apoptosis in mtNSC-34 cells. a Overexpressed miR-18b (miR-18b-5p) decreased Hif1 alpha & Mef2c proteins. Both Mctp1 & Rarb expression were increased by miR-18b (miR-18b-5p). Downregulated Bax & upregulated Bcl2 by miR-18b (miR-18b-5p) diminished apoptosis in mtNSC-34 cells. b RT-qPCR analysis showed low expression of Hif1 alpha & Mef2c mRNAs. c Mctp1 & Rarb transcripts were highly expressed by miR-18b (miR-18b-5p). d Bax mRNAs were decreased & Bcl2 mRNAs were increased by overexpressed miR-18b. e miR-18b (miR-18b-5p) was overexpressed in mtNSC-34 cells. f miR-206 was reduced by miR-18b (miR-18b-5p). g LDH release analysis explained that transfected miR-18b (miR-18b-5p) restores apoptosis. h Luciferases assay with 3′ UTR of Hif1 alpha showed that Hif1 alpha is target of miR-18b in contNSC-34 cells. i & j Overexpression of miR-18b (miR-18b-5p) enhanced neuronal differentiation (MAP2) & attenuated intracellular Ca2+ levels (Cont (0.098) versus miR-18b (miR-18b-5p) (0.051) in fluorescence intensities from baseline 490/525 ratio) in mtNSC-34 cells. Empty vector served as a negative control (Cont). Arrow represents SOD1 aggregation (green). Scale bar, 40 μm. Significantly different at *, p < 0.05; **, p < 0.005. The experiments were replicated 5 times Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/32605607), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: HIF-1 alpha Antibody [NB100-449] - Hypoxic conditions upregulate CNN3 in BeWo cells.BeWo cells were cultured either under normoxic or under hypoxic conditions for 24(A) Total protein lysates were examined with the Western Blot technique to detect CNN3 protein levels. For normalization, HPRT was stained on the same membrane. As hypoxia marker, HIF-1 alpha was detected as well. (B) Protein bands of CNN3 & HPRT were densitometrically measured on the Western Blot membrane & CNN3/HPRT levels are plotted in the graph (white column: normoxia; black column: hypoxia). n = 3. (C) BeWo cells were serum starved for 16 h & then treated with either PBS as control or 200 µM CoCl2 for 6 h in serum free medium. Then total protein was isolated & a Western Blot was performed. The HIF-1 alpha, CNN3 & HPRT protein was detected on the membrane with specific antibodies. (D) A densitometric analysis was performed to determine CNN3/HPRT levels (white column: normoxia; black column: hypoxia). n = 3. Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/25050546), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: HIF-1 alpha Antibody [NB100-449] - Dynamics of the hypoxia-triggered decrease in MET phosphorylation & its reversal upon reoxygenation(A) MCF10A cells were incubated under normoxia or hypoxia for 1 h. They were then treated, under the same oxygen pressure, with 10 ng/mL HGF/SF for 5, 10, 20, 30, 60, 90 & 120 minutes. A control (Ctrl) without any HGF/SF stimulation was also performed. The same amount of protein was analyzed by western blotting with antibodies directed against: phosphorylated residues in the MET kinase domain, the MET kinase domain, the hypoxia marker HIF1a, phosphorylated Akt, Akt, Erk2, phosphorylated Erk, & actine. The positions of prestained molecular weight markers are indicated. Arrows indicate the positions of precursor & mature full-length MET. (B) MCF10A cells were incubated under hypoxia for 5, 10, 15 or 30 minutes. Another set of cells were incubated under hypoxia for 1 h & then returned to normoxia for 5, 10, 15 or 30 min (re-oxygenation). A control under normoxic (N) conditions was also included. The same amount of protein was analyzed by western blotting as previously described with the addition of GAB1 & its phosphorylated form. (C) MCF10A cells were placed under normoxic or hypoxic conditions for 1 h or hypoxia for 1 h & then normoxia for 10 minutes (reoxygenation). Cells were then treated at the indicated time with 10 ng/mL of HGF/SF. Cell lysates were incubated for AlphaScreen specific phospho-Erk & phospho-Akt quantitation. Error bars represent standard deviations (n = 3; ± SD). Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/29930749), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: HIF-1 alpha Antibody [NB100-449] - The role of HIF-1 in purinosome formation.a, quantifying the number of purinosome-containing cells in normoxia or hypoxia (24 h) in purine-rich medium & normoxia in purine-depleted medium (purine -ve), cells in hypoxia transfected with siRNA to HIF-1 alpha (+ siRNA), & cells in purine-rich medium supplemented with DFX. Data shown are n = 3, mean ± S.E., total number of cells counted are shown in parentheses. b, time course of purinosome formation in hypoxia shows the number of purinosome-containing cells steadily increases after 3 h in hypoxia. Re-oxygenation of the samples after hypoxic incubation for 10 h reverts the number purinosome-containing cells back to normoxic levels. Data shown is n = 3, mean ± S.E., total number of cells counted are shown in parentheses. c, time course of HIF-1 alpha stabilization in hypoxia shows maximum HIF-1 alpha protein expression levels at 3 h in hypoxia, after which the HIF-1 alpha expression decreases. The positions of molecular markers are shown for each blot; uncropped blots with overlaid markers are deposited in the raw data files. d, the effect of hypoxia on the transcription of purine biosynthesis enzymes measured by qPCR. Vascular endothelial growth factor (VEGF) & HIF-1 alpha are controls. Data shown are n = 5, mean ± S.E. e, the effect of hypoxia on the protein expression levels of the purine biosynthetic enzymes. HIF-1 alpha is stabilized in hypoxia as expected, & no significant increase in the purine enzymes was detected between normoxic (21% oxygen) & hypoxic (1% oxygen) growth conditions. The positions of molecular markers surrounding each band of interest are shown for each blot; uncropped blots with overlaid markers are deposited in the raw data files. Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/32439803), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: HIF-1 alpha Antibody [NB100-449] - C/EBPδ promotes HIF-1 alpha expression in macrophages through inhibition of FBXW7 alpha a) RT-PCR analysis of FBXW7 isoform expression from different sources as follows. 1, primary peritoneal macrophages (PPMs); 2, RAW 264.7 cells; 3, MMTV-Neu mammary tumour tissue; 4, primary mouse embryo fibroblasts. Numbers indicate the position of size markers in base pairs. (b) RT-qPCR analysis of Fbxw7 transcript levels in PPMs from WT & Cebpd−/− KO mice, cultured +/− LPS (100 ng/ml, 24 h), compared to WT untreated (n=4, *P<0.05; **P<0.001). (c) Western analysis of nuclear extract (NE) from primary human monocytes nucleofected with siRNA oligos (C, control; D, CEBPD; F, FBXW7) & treated with LPS (100 ng/ml) & 1%O2 (16 h) as indicated. SE, short exposure; LE, long exposure. (d) RT-qPCR analysis of FBXW7 & CEBPD transcripts in primary human monocytes as in panel (c) (n=3, *P<0.05; **P<0.001). (e) Western analysis of NE from PPMs nucelofected with siRNA oligos & treated with LPS (100 ng/ml) & 1%O2 for 16 h as indicated. SE, short exposure; LE, long exposure.Where applicable, data are mean±S.E.M., evaluated by two-tailed unequal variance t-test. Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/23575666), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: HIF-1 alpha Antibody [NB100-449] - Hypoxia downregulates CFTR expression. (a) Phase-contrast photographs of MDCK cells treated with normoxia & hypoxia conditions; (b) Western blot showing downregulation of CFTR in the MDCK cells by hypoxia (Full-length blot is shown in Supplementary Figure S7b.); (c) Real time-PCR assay showing decreased mRNA expression of CFTR induced by hypoxia in HK-2 cells,*p < 0.05; (d) Western blot showing decreased expression of CFTR induced by hypoxia in HK-2 cells (Full-length blot is shown in Supplementary Figure S7c.); (e) Western blot showing the expression changes of HIF-1 alpha & EMT markers induced by hypoxia in MDCK cells, quantification analysis is shown in the lower panel, *p < 0.05; (Full-length blot is shown in Supplementary Figure S7d.) (f) Immunofluorescent staining showing dramatically increased HIF-1 alpha & reduced CFTR protein levels in tubular epithelial cells at inner cortices in UUO kidney (arrow). Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/28701694), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: HIF-1 alpha Antibody [NB100-449] - NRP1 & MET on HT1080 cells are accessible for blood-borne rhodocetin-alpha beta (A) expression of NRP1 & MET by HT1080 cells was proven by flow cytometry. Gray, isotype-matched controls. (B) flow cytometry of NRP1-knockout HT1080 cells demonstrating their NRP1-deficiency & unaffected MET expression. (C) treatment with CoCl2, mimicking a hypoxic tumor micro-environment, induced upregulation of HIF-1 alpha . (D) NRP1, as a downstream target of HIF-1 alpha , is upregulated in HT1080 cells but not in NRP1-knockout HT1080 cells. beta -actin immunoblots show even loading. (E) increased HIF-1 alpha (red) levels in hypoxic tumor regions, which also contained partly (arrows) or completely (open arrows) EC-deficient VM vessels. ECs are stained green & nuclei blue. (F) immunostaining of NRP1 (red) & MET (blue) showed that both proteins were present on HT1080 cells & ECs in tumor tissue. Note the continuity between EC-lined vasculature & EC marker-deficient vessels (arrows in F’). F’, F’’, in oblique view, gating of the green CD31 signal also showed an apical absence of NRP1 on ECs in contrast to MET (open arrows). (G) the fluorescence intensity along a traceroute, averaged over a width of 5 pixels, (rectangle in F) through the endothelium revealed that in ECs NRP1, unlike MET, is absent from the apical side & restricted to the basolateral side. In contrast, on ATV/VM-lining cells (F’, arrows) both NRP1 & MET are accessible from the bloodstream (H). Vertical gray lines in G & H indicate the position of the apical cell border. Original magnification was 400× (E) & 630× (F-F’’). Representative images are shown. Image collected & cropped by CiteAb from the following publication (//www.oncotarget.com/lookup/doi/10.18632/oncotarget.25032), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Immunohistochemistry: HIF-1 alpha Antibody [NB100-449] - NRP1 & MET on HT1080 cells are accessible for blood-borne rhodocetin-alpha beta (A) expression of NRP1 & MET by HT1080 cells was proven by flow cytometry. Gray, isotype-matched controls. (B) flow cytometry of NRP1-knockout HT1080 cells demonstrating their NRP1-deficiency & unaffected MET expression. (C) treatment with CoCl2, mimicking a hypoxic tumor micro-environment, induced upregulation of HIF-1 alpha . (D) NRP1, as a downstream target of HIF-1 alpha , is upregulated in HT1080 cells but not in NRP1-knockout HT1080 cells. beta -actin immunoblots show even loading. (E) increased HIF-1 alpha (red) levels in hypoxic tumor regions, which also contained partly (arrows) or completely (open arrows) EC-deficient VM vessels. ECs are stained green & nuclei blue. (F) immunostaining of NRP1 (red) & MET (blue) showed that both proteins were present on HT1080 cells & ECs in tumor tissue. Note the continuity between EC-lined vasculature & EC marker-deficient vessels (arrows in F’). F’, F’’, in oblique view, gating of the green CD31 signal also showed an apical absence of NRP1 on ECs in contrast to MET (open arrows). (G) the fluorescence intensity along a traceroute, averaged over a width of 5 pixels, (rectangle in F) through the endothelium revealed that in ECs NRP1, unlike MET, is absent from the apical side & restricted to the basolateral side. In contrast, on ATV/VM-lining cells (F’, arrows) both NRP1 & MET are accessible from the bloodstream (H). Vertical gray lines in G & H indicate the position of the apical cell border. Original magnification was 400× (E) & 630× (F-F’’). Representative images are shown. Image collected & cropped by CiteAb from the following publication (//www.oncotarget.com/lookup/doi/10.18632/oncotarget.25032), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: HIF-1 alpha Antibody [NB100-449] - Dectin-1-induced TSLP negatively regulates pro-IL-1 beta & HIF-1 alpha . (A) Human mDC were stimulated with SC glucan, CA glucan or heat killed C. albicans hyphae with anti-TSLPR antibodies or IgG isotype control for 24 h (n = 6 independent donors, presented as pooled data). Lactate production was measured in cell-culture supernatants using colourmetric L-lactate detection kit. (B) Human mDC were stimulated SC glucan with either anti-TSLP, anti-TSLPR or IgG isotype control antibodies for 8 h (n = 1 representative donor presented, three separate experiments performed). Pro-IL-1 beta , IL-1 beta , HIF-1 alpha , phospho-p38 MAPK, p38 MAPK, phospho-AMPK, AMPK & beta -actin were measured by immunoblot. (C–G) Densitometry of cumulative data was performed using Image Studio Lite software with pro-IL-1 beta , IL-1 beta & HIF-1 alpha normalized to beta -actin & phospho-p38 MAPK & phospho-AMPK normalized to total p38 MAPK & AMPK respectively. Data is reported as percentage of maximal signal observed within each donor (n = 3 independent donors, presented as pooled data). Cumulative data displayed as mean +SEM. Statistical analysis calculated using one-way ANOVA with Bonferroni post-tests (***p = 0.001). Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/31139177), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: HIF-1 alpha Antibody [NB100-449] - Involvement of MET in Akt & Erk pathway activation under hypoxiaMCF10A cells were transfected with a pool of three MET-targeting siRNAs (20 nM) or a control siRNA (siCtrl). A control without siRNA was also included (Ctrl) (A). MCF10A cells were transfected with two HIF1a-targeting siRNAs (20 nM), independently or together, or a control siRNA (siCtrl) (B). The cells were then placed for 1 h under normoxic or hypoxic conditions & treated or not for 10 min with 10 ng/mL HGF/SF. In each experiment, the same amount of protein was analyzed by western blotting with antibodies directed against: phosphorylated residues in the MET kinase domain, the MET kinase domain, phosphorylated Akt, Akt, phosphorylated Erk, Erk2, phosphorylated GAB1, GAB1, or hypoxia marker HIF1a. The positions of prestained molecular weight markers are indicated. Arrows indicate the positions of precursor & mature full-length MET & Erk1/2 proteins. Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/29930749), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: HIF-1 alpha Antibody [NB100-449] - beta -Glucan size affects the requirement for reactive oxygen species (ROS) in IL-1 beta induction. (A) Human monocyte-derived dendritic cell (mDC) stimulated with curdlan or glucan-mp for 8 h. ROS were detected by incubating cells with CellRox Green fluorescence dye & analysis by flow cytometry (representative experiment of two) presented, (B) Human mDCs from healthy donors or chronic granulomatous disease (CGD) patients were stimulated with curdlan or glucan-mp for 8 h. HIF-1 alpha & pro-IL-1 beta protein expression measured by immunoblot (n = 2 donors). (C–L) Human mDC from healthy donors or CGD patients were stimulated with curdlan or glucan-mp for 24 h (n = 6 donors). IL-1 beta , IL-6, IL-23, TSLP, & CCL22 secretion were measured by enzyme-linked immunosorbent assay, with cumulative data displayed as mean ± SEM. Image collected & cropped by CiteAb from the following publication (//journal.frontiersin.org/article/10.3389/fimmu.2017.00791/full), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Reactivity:

Hu, Mu, Rt, Ca, Ch, Gt, Pm, Pm, Bv

Species Glossary

Reactivity: Hu, Mu, Rt, Ca, Ch, Gt, Pm, Pm, Bv

Applications:

, , , , , , , , ,

Applications: , , , , , , , , ,

Host:

Rabbit Polyclonal

Host: Rabbit Polyclonal

Sizes:

Sample Size Available

Sizes: Sample Size Available

Conjugate:

Unconjugated

Validated by:

Validated by:

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NB100-134

HIF-1 alpha Antibody - BSA Free

(14 Reviews)

(227 Publications)

0.1 ml

NB100-134

Immunohistochemistry-Paraffin: HIF-1 alpha Antibody - BSA Free [NB100-134] - Analysis of HIF-1 alpha in zebrafish retina tissue using NB100-134 HIF-1 alpha antibody. Mouse tissue was used as a control. Image from verified customer review.

Western Blot: HIF-1 alpha Antibody - BSA Free [NB100-134] - Analysis of HIF-1 alpha in overexpression and endogenous HIF-1 alpha & HIF-2 alpha using anti-HIF-1 alpha antibody NB100-134. The data showed that HIF-1 alpha antibody did not react to HIF-2 alpha overexpression. Image from verified customer review.

Western Blot: HIF-1 alpha Antibody - BSA Free [NB100-134] - Analysis of HIF-1 alpha using NB100-134, on normoxic and hypoxic nuclear rat cell lysates.

<b>Biological Strategies Validation. </b>Immunocytochemistry/Immunofluorescence: HIF-1 alpha Antibody - BSA Free [NB100-134] - Representative images of macrophages and activated macrophages, in the absence or presence of 10 uM hemin for 72 h. HIF-1 alpha levels were detected by immunofluorescence using a confocal imaging system (Opera Phenix, Perkin Elmer). Images were acquired with 20x water objective. Staining intensity levels in the nucleus and cytosolic region were obtained using Harmony software (Perkin Elmer). Nucleus and cytosol were identified through Hoechst (at 405 nm) and CellMask (at 655 nm) staining, respectively. Scale is shown as 100 um. Image collected and cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/33187129/) licensed under a CC-BY license.

Immunocytochemistry/Immunofluorescence: HIF-1 alpha Antibody - BSA Free [NB100-134] - Analysis of HIF-1 alpha in Normoxic (N) and Hypoxic (H) ARPE-19 cells using anti-HIF-1 alpha antibody NB100-134. Image from verified customer review.

Immunocytochemistry/Immunofluorescence: HIF-1 alpha Antibody - BSA Free [NB100-134] - Hif1alpha expression in 9.5dpc primordial germ cells. Whole-mount staining of Hif1alpha and GFP in Oct4-GFP embryo (lateral view). Scale bar: 100um. Image collected and cropped by CiteAb from the following publication (//dx.plos.org/10.1371/journal.pone.0154309) licensed under a CC-BY license.

Immunohistochemistry-Paraffin: HIF-1 alpha Antibody - BSA Free [NB100-134] - Staining in the canine CL on days 10 to 70 after ovulation. PC = positive control (human placenta). NC = negative control. Image from verified customer review.

Immunohistochemistry: HIF-1 alpha Antibody - BSA Free [NB100-134] - Staining of human kidney, renal tubular epithelium in cortex using NB100-134.

Immunohistochemistry-Paraffin: HIF-1 alpha Antibody - BSA Free [NB100-134] - Analysis of FFPE tissue section of human endometrium carcinoma AN3CA cell line based xenograft using rabbit polyclonal HIF-1 alpha antibody NB100-134 at 1:300 dilution. The signal was developed using HRP-labelled secondary antibody and DAB reagent, and the section was further counterstained using hematoxylin. The tested section depicted mainly a diffused cytoplasmic staining but there were some cells which showed nuclear signal also (representing hypoxic cells).

Simple Western: HIF-1 alpha Antibody - BSA Free [NB100-134] - Image shows a specific band for HIF-1 alpha in 0.2 mg/mL of Hypoxic HeLa lysate. This experiment was performed under reducing conditions using the 12-230 kDa separation system.

Immunoprecipitation: HIF-1 alpha Antibody - BSA Free [NB100-134] - PC-3 cell lysates run with cobalt chloride treatments. Immunoprecipitation was performed with protein A/G agarose beads. This data was provided courtesy of Kelie Reece, Figg lab, NCI.

Dual RNAscope ISH-IHC: HIF-1 alpha Antibody - BSA Free [NB100-134] - Formalin-fixed paraffin-embedded tissue sections of human kidney were probed for HIF-1 alpha mRNA (ACD RNAScope Probe, catalog #605228; Fast Red chromogen, ACD catalog # 322750). Adjacent tissue section was processed for immunohistochemistry using Rabbit Polyclonal (Novus Biologicals catalog # NB100-134) at 1:500 dilution with one-hour incubation at room temperature followed by incubation with anti-rabbit IgG VisUCyte HRP Polymer Antibody (Catalog # VC003) and DAB chromogen (yellow-brown). Tissue was counterstained with hematoxylin (blue). Specific staining was localized to cytoplasm of tubules.

Simple Western: HIF-1 alpha Antibody - BSA Free [NB100-134] - Simple Western lane view shows lysates of MCF +/- DMOG, BioSpherix MSCs in hypoxic conditions, and BioSpherix MSCs in normoxic conditions loaded at 0.5 mg/ml. A band was detected for HIF-1 alpha at approximately 116 kDa (as indicated) using NB100-134 (1:100 dilution) followed by Anti-Rabbit Secondary Antibody (042-206, ProteinSimple). This experiment was conducted under standard assay conditions, and using the 12-230 kDa separation module (SW-W004). Non-specific interaction with the 230 kDa Simple Western standard may be seen with this antibody. Image from an internal validation.

P53 was increased response to IR in the fetal brain.Even though no significant change in protein level, total p53 and phosphorylation of P53 at ser15 were increased compared with HIF-1 alpha in fetal brains response to IR. Numbers in the pictures show the average of three independent experiments (n = 3 from 3 individual pregnant mice).

WNT11 is induced by hypoxia or hypoxic mimetics in different cell types.(A) Increased Wnt11 mRNA in EMSC adipocytes (Day 12) after hypoxia-mimetic treatments. EMSC adipocytes were treated with CoCl2 (0.1 mM), DFO (0.1 mM) or DMOG (0.1 mM) for 24 hrs. Values were normalized to Tbp mRNA & are expressed relative to control (n = 3). (B,C) Increased Wnt11 mRNA by hypoxia in EMSC preadipocytes & adipocytes (Day 0–12 after differentiation) (B), & C2C12 myoblast & myocyte (Day 0 & 8 after differentiation) (C). Wnt11 mRNA was assessed by quantitative PCR in cells exposed to air (21% O2) or hypoxia (1% O2) for 24 hrs. (n = 4). Values were normalized to Tbp mRNA & are expressed relative to 21% O2 samples (left panel). (D) Immunoblot analyses of HeLa cells under normal air or hypoxia for 24 hrs. (E,F) Induction of Wnt11 by increasing concentrations of DMOG in MDA-MB-231 cells (E) & 4T1 cells (F). (G) EMSCs treated with 0.1 mM DMOG for the indicated times. Wnt11 & Vegf mRNA expression was measured by qPCR & normalized to Tbp mRNA (n = 4). (H) WNT11 protein levels after DMOG treatment normalized to alpha -Tubulin (upper panel; n = 4). Representative immunoblots of EMSCs treated with 0.1 mM DMOG for the indicated times (Lower panel). (I) Protein expression in MDA-MB-231 cells treated with 0.1 mM DMOG. (J) Induction of Wnt11 promoter activity by hypoxia or hypoxia mimetics. pGL3-Wnt11 promoter plasmid was transfected into C2C12 cells. Cells were incubated with DMOG (left panel, n = 4) or under 21% O2 or 1% O2 (right panel, n = 8) for 24 hrs. For panels (A–C,G,H,J), values are mean ± s.e.m. *p 25 pictures/group from three separate experiments. Scale bar = 40 µm. (F) Cell proliferation was assessed by evaluating PCNA expression, n = 4. * p 20). B May–Grunwald–Giemsa staining of whole KM blood cells in 6-month-old AB zebrafish after 4 days of E2 treatment (t-test, ***p 20). F Effect of E2 on neutrophil proliferation in AB zebrafish embryos. (one-way ANOVA (LSD). ns, no significance, n > 10). G E2 promotes the apoptosis of myeloid lineage cells in AB zebrafish embryos (one-way ANOVA (LSD) **p 10). Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/35842445), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: HIF-1 alpha Antibody - BSA Free [NB100-134] - IGFBP5 inhibits HIF1 alpha expression through the MAPK-ERK signaling pathwayA. & B. Western blots using specific antibodies for the phosphorylation state of IGF1R, ERK1/2, & p38-MAPK from A375 IGFBP5 OE & control cells. Phosphorylation of IGF1R, ERK1/2, & p38-MAPK was decreased in A375 cells transfected with IGFBP5. GAPDH was used as a loading control. C. Overexpression of IGFBP5 inhibited HIF1 alpha expression visualized by western blots of A375 cells. The arrow points to the band of HIF1 alpha . D. Assessment of the reduced gene expression levels of VEGFA & MMP9, downstream genes regulated by HIF1 alpha , in A375 IGFBP5 OE compared to control cells by qRT-PCR analysis. Data were shown for the mean ± SD form three independent experiments. P values based on two-side Student t-test comparing A375 IGFBP5 OE tumor cells & vector control cells. *, P < 0.05. Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/26010068), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: HIF-1 alpha Antibody - BSA Free [NB100-134] - WNT11 regulates MMPs activities.(A–C) (Top panels): Serum-free medium was conditioned for 24 hrs by the indicated cells, concentrated 20-fold & assayed by gelatin zymography. Gelatinolytic activity is indicated by clear zones against a dark background of stained substrate. (Bottom): Whole cell extracts were immunoblotted with indicated antibodies. (A) Overexpression of Wnt11 in EMSC or BT473 cells enhances activity of MMP-9 & MMP-2. (B) Impaired activity of MMP-9 & MMP-2 in MDA-MB-231 cells (left) or EMSCs (right) stably expressing Wnt11 shRNAs & treated with DMOG. (C) WNT11 is required for MMP-9 & MMP-2 activity in MDA-MB-231 cells (left) or EMSCs (right) under normoxic & hypoxic culture conditions. (D) WNT11 regulates MMP2 protein in media. (Top): conditioned media from indicated cells & treatments. (Bottom): whole cell lysates were immunoblotted with indicated antibodies. (E) Recombinant WNT11 induces both MMP-2 protein & MMP-2 activity in media. (Top panels): Gelatin zymography & immunoblot of serum-free medium conditioned for the indicated times after recombinant WNT11 (r-WNT11) treatment. (Bottom): Whole cell lysates were immunoblotted with indicated antibodies. (F) MMP-2 inhibitor attenuated induced migration by WNT11. MDA-MB-231 cells infected with lentiviruses for stable expression of Wnt11 or GFP (n = 4) were incubated with either vehicle or 1 μM of ARP100. Media in the lower compartment had same concentration of DMSO or inhibitor. Values are mean ± s.e.m. *p < 0.05, **p < 0.01. For panels (A–D), HIF-1 alpha & HIF-2 alpha were shown as a marker of hypoxia, WNT11 normalized to alpha -Tubulin was shown. Image collected & cropped by CiteAb from the following publication (//www.nature.com/articles/srep21520), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: HIF-1 alpha Antibody - BSA Free [NB100-134] - ER-INP binds to PHD2, inhibits hydroxylation of HIF-1 alpha & increases HIF-1 alpha accumulation. (A) Co-immunoprecipitation assay. HEK293 cells were transfected with control vector or intrabody ER-INP for 48 hours & subsequently lysed. Co-immunoprecipitation & western blot assays were performed on the cell lysis. ER-INP recognized & bound to PHD2 in HEK293 cells (n = 3). (B) Western blot analysis to measure the effect of ER-INP on HIF-1 alpha & its hydroxylation level in transfected HEK293 & RAW264.7 cells pre-treated with MG132 (upper panel) & the protein ratio to beta -actin loading control by ImageJ densitometry analysis (lower panel; n = 3). (C) Immunofluorescence assay. Expression of ER-INP increases HIF-1 alpha accumulation in RAW264.7 cells. Cells were stained with anti-HIF-1 alpha antibody & DAPI & then visualized & photographed under immunofluorescence microscopy (left), & mean fluorescence intensity of HIF-1 alpha versus the mean fluorescence intensity of nuclear DAPI staining is displayed (right). Data represent the mean ± SD of 3 independent slides. Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/31413262), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: HIF-1 alpha Antibody - BSA Free [NB100-134] - HIF-1 alpha binds to TIM-3 promoter & regulates its expression in primary glia.(a) Cell surface expression of TIM-3 analysed in BV2 cells under 20% O2 or 1% O2 for 24 h by flow cytometry using PE-conjugated anti-TIM-3 antibody. Results from 3 independent experiments presented as a representative histogram & the mean fold change (± s.d.) relative to normoxic sample. (b) Mouse primary mixed glial cells incubated under hypoxia or normoxia for 24 h, & the cells examined by immunocytochemistry using an anti-TIM-3 antibody. (c,d) Mouse primary mixed glial cells & primary neuronal cells incubated under hypoxia or normoxia for 24 h, & then RT–PCR used to detect the levels of TIM-3 & actin. Relative transcript levels shown as the mean fold change (± s.d.) from 3 independent experiments (NS, not significant, Student–Newman–Keuls test). (e) Primary mixed glial cells incubated under hypoxia or normoxia for 24 h, & chromatin immunoprecipitation (ChIP) performed w/ anti-HIF-1 alpha or control IgG. Results presented as relative amounts representative of 3 independent experiments. (f) Primary mixed glial cells cultured from HIF-1 alpha +f/+f mice, infected w/ Ad-GFP or Ad-Cre/GFP, transfected w/ TIM-3-luciferase reporter constructs & incubated under hypoxic or normoxic conditions for 24 h. Relative promoter activity is expressed as the ratio of luciferase activity/ beta -galactosidase activity. (g,h) RT–PCR (g) & WB analysis (h) performed under hypoxia or normoxia for 24 h using the indicated primers & antibodies, respectively. The data shown representative of at least 3 independent experiments. The graphs show the % changes in TIM-3 transcript & protein levels in Ad-Cre/GFP- versus Ad-GFP-infected cells under hypoxia. IP, immunoprecipitation. Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/25790768), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: HIF-1 alpha Antibody - BSA Free [NB100-134] - Regulation of catabolic genes by HIF-1 alpha . (A) Protein levels of Hif1a, & mRNA levels of Mmp13, & Hif2a in WT mouse primary chondrocytes transduced with GFP or HIF1A adenoviral vectors under the hypoxic condition (3% O2). GFP or HIF1A was transduced at a multiplicity of infection (MOI) of 100. The cells were treated with or without 10 ng/mL IL-1 beta for 2 days. Bars show the mean ± SD of three samples per group. *P < 0.05. (B) Protein levels of Hif1a, & mRNA levels of Mmp13, & Hif2a in WT mouse primary chondrocytes transfected with siRNA against GFP or Hif1a under the hypoxic condition. The cells were treated with or without 10 ng/mL IL-1 beta for 2 days. Bars show the mean ± SD of three samples per group. *P < 0.05. (C) Protein levels of Hif1a, & mRNA levels of Mmp13, & Hif2a in Hif1afl/fl primary chondrocytes transduced with GFP or Cre adenoviral vectors under the hypoxic condition. GFP or Cre was transduced at a MOI of 100. The cells were treated with or without 10 ng/mL IL-1 beta for 2 days. Bars show the mean ± SD of three samples per group. *P < 0.05. Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/32214220), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: HIF-1 alpha Antibody - BSA Free [NB100-134] - NSCs Reduce Succinate Levels & Reprogram the Metabolism of Type 1 Pro-inflammatory Mφ toward Oxidative Phosphorylation In Vitro(A) Experimental setup for in vitro MφLPS co-cultures with iNSCs/NSCs.(B & C) Gene expression microarrays of MφLPS-iNSCs/NSCs. (B) Venn diagram of differentially expressed genes (adjusted p value < 0.1). (C) Heatmap of genes differentially expressed (adjusted p value < 0.1) in MφLPS-iNSCs or MφLPS-NSCs.(D & E) qRT-PCR independent validation of differentially expressed inflammatory genes as in (C). (D) Expression of genes related to type 1 inflammatory (E) & anti-inflammatory Mφ phenotypes relative to Actb. Data are mean fold change (±SEM) versus MφLPS from n ≥ 3 independent replicates per condition.(F) qRT-PCR of BV2LPS-iNSCs/NSCs (±SEM) from n ≥ 3 independent experiments per condition. BV2 & BV2LPS are shown as controls.(G & H) Extracellular flux (XF) assay of the oxygen consumption rate (OCR) (G) & extracellular acidification rate (ECAR) (H) in MφLPS-iNSCs/NSCs. Data were normalized on total protein content & are expressed as mean values (±SEM) from n ≥ 3 independent experiments per condition.(I & J) Levels of significantly changed extracellular (EXTRA_Metab, I) & intracellular (INTRA_Metab, J) metabolites in MφLPS versus Mφ at 25 hr. Data are mean a.u. (±SEM) from n ≥ 2 independent experiments per condition.(K & L) Hif-1 alpha (K), PKM2 (K), & IL-1 beta (L) expression levels relative to beta -actin. Data are mean fold change versus MφLPS (±SEM) from n ≥ 3 independent experiments per condition.∗p ≤ 0.05 & ∗∗p ≤ 0.01 versus MφLPS. See also Tables S2 & S3. Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/29478844), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: HIF-1 alpha Antibody - BSA Free [NB100-134] - Enhanced expression of hypoxia-induced survival factors & angiogenic growth factors in hASC L-spheroids.(A) The light source used was LED (660 nm) designed to fit over a microplate (12.5 × 8.5 cm) for cell culture. (B) Formation of hASC L-spheroids. hASCs morphology on non–tissue culture–treated 24-well plates at day 3. Scale bar = 500 μm. (C) Western blot analysis & quantification of HIF1-alpha in hASCs cultured as spheroids, L-spheroids & monolayers (*p < 0.01, compared to the L-spheroid group). (D) Angiogenesis-related protein analysis of L-spheroids (*, p < 0.05, compared to the spheroid group, t-test, n = 3 in each group). (E) ELISA measurement of spheroids cultured for 3 days. Concentrations of VEGF are presented as pg-corrected for 104 cells. (*, p < 0.05, compared with spheroid 6J/cm2 group, t-test, n = 3 in each group). Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/26065900), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Reactivity:

Hu, Mu, Rt, Bv, Ca, Gp, Pm, Xp, Ze

Species Glossary

Reactivity: Hu, Mu, Rt, Bv, Ca, Gp, Pm, Xp, Ze

Applications:

, , , , , , , , , ,

Applications: , , , , , , , , , ,

Host:

Rabbit Polyclonal

Host: Rabbit Polyclonal

Sizes:

Sample Size Available

Sizes: Sample Size Available

Conjugate:

Unconjugated

Available Conjugates:

Validated by:

Validated by:

Conjugate	Catalog #	Availability	Size	Price
Alexa Fluor 350	NB100-134AF350
Alexa Fluor 405	NB100-134AF405
Alexa Fluor 488	NB100-134AF488
Alexa Fluor 532	NB100-134AF532
Alexa Fluor 594	NB100-134AF594
Alexa Fluor 647	NB100-134AF647
Alexa Fluor 700	NB100-134AF700
Alexa Fluor 750	NB100-134AF750
Allophycocyanin	NB100-134APC
Biotin	NB100-134B
CoraFluor 1	NB100-134CL1
DyLight 350	NB100-134UV
DyLight 405	NB100-134V
DyLight 488	NB100-134G
DyLight 550	NB100-134R
DyLight 594	NB100-134DL594
DyLight 650	NB100-134C
DyLight 680	NB100-134FR
DyLight 755	NB100-134IR
FITC	NB100-134F
HRP	NB100-134H
Janelia Fluor 525	NB100-134JF525
Janelia Fluor 549	NB100-134JF549
Janelia Fluor 646	NB100-134JF646
Janelia Fluor 669	NB100-134JF669
PE	NB100-134PE
PerCP	NB100-134PCP
mFluor Violet 450 SE	NB100-134MFV450
mFluor Violet 500 SE	NB100-134MFV500
mFluor Violet 610 SE	NB100-134MFV610

Available Formulations

Formulation	Catalog #	Availability	Price
BSA Free	NB100-134SS

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NBP2-75977

HIF-1 alpha Antibody (2443B) - BSA Free

0.1 mg

NBP2-75977

Immunocytochemistry/Immunofluorescence: HIF-1 alpha Antibody (2443B) [NBP2-75977] - Immunofluorescent staining of HIF-1 alpha using catalog number NBP2-75977 (clone 2443B). Staining was observed in Hela cells treated with DFO but not in untreated Hela cells.

Simple Western: HIF-1 alpha Antibody (2443B) [NBP2-75977] - Lane view shows a specific band for HIF-1 alpha in HepG2 +/- DFO cell lysate using 20ug/ml. Electropherogram image of corresponding Simple Western lane view at WES molecular weight of 116 KDa. Image from an internal validation.

Simple Western: HIF-1 alpha Antibody (2443B) [NBP2-75977] - Simple Western lane view shows lysates of MCF +/- DMOG, BioSpherix MSCs in hypoxic conditions, and BioSpherix MSCs in normoxic conditions loaded at 0.5 mg/ml. A band was detected for HIF-1 alpha at approximately 116 kDa (as indicated) using NBP2-75977 (1:100 dilution) followed by Anti-Rabbit Secondary Antibody (042-206, ProteinSimple). This experiment was conducted under standard assay conditions, and using the 12-230 kDa separation module (SW-W004). Non-specific interaction with the 230 kDa Simple Western standard may be seen with this antibody. Image from an internal validation.

Reactivity:

Hu

Species Glossary

Reactivity: Hu

Applications:

, , ,

Applications: , , ,

Host:

Rabbit Monoclonal

Host: Rabbit Monoclonal

Sizes:

Sample Size Available

Sizes: Sample Size Available

Conjugate:

Unconjugated

Available Conjugates:

Validated by:

Validated by:

Conjugate	Catalog #	Availability	Size	Price
Alexa Fluor 350	NBP2-75977AF350
Alexa Fluor 405	NBP2-75977AF405
Alexa Fluor 488	NBP2-75977AF488
Alexa Fluor 532	NBP2-75977AF532
Alexa Fluor 594	NBP2-75977AF594
Alexa Fluor 647	NBP2-75977AF647
Alexa Fluor 700	NBP2-75977AF700
Alexa Fluor 750	NBP2-75977AF750
Allophycocyanin	NBP2-75977APC
Biotin	NBP2-75977B
CoraFluor 1	NBP2-75977CL1
DyLight 350	NBP2-75977UV
DyLight 405	NBP2-75977V
DyLight 488	NBP2-75977G
DyLight 550	NBP2-75977R
DyLight 594	NBP2-75977DL594
DyLight 650	NBP2-75977C
DyLight 680	NBP2-75977FR
DyLight 755	NBP2-75977IR
FITC	NBP2-75977F
HRP	NBP2-75977H
Janelia Fluor 525	NBP2-75977JF525
Janelia Fluor 549	NBP2-75977JF549
Janelia Fluor 646	NBP2-75977JF646
Janelia Fluor 669	NBP2-75977JF669
PE	NBP2-75977PE
PerCP	NBP2-75977PCP
mFluor Violet 450 SE	NBP2-75977MFV450
mFluor Violet 500 SE	NBP2-75977MFV500
mFluor Violet 610 SE	NBP2-75977MFV610

Available Formulations

Formulation	Catalog #	Availability	Price
BSA Free	NBP2-75977SS
Azide and BSA Free	NBP2-80764

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NBP2-75978

HIF-1 alpha Antibody (2443C) - BSA Free

(2 Reviews)

(1 Publication)

0.1 mg

NBP2-75978

Immunocytochemistry/Immunofluorescence: HIF-1 alpha Antibody (2443C) [NBP2-75978] - Immunofluorescent staining of HIF-1 alpha using catalog number NBP2-75977 (clone 2443B). Staining was observed in Hela cells treated with DFO but not in untreated Hela cells.

Immunohistochemistry-Paraffin: HIF-1 alpha Antibody (2443C) [NBP2-75978] - HIF-1 alpha (clone 2443C) was detected in immersion fixed paraffin-embedded sections of human kidney using Rabbit Anti-Human HIF-1 alpha Monoclonal Antibody (Catalog # NBP2-75978) at 1.0 ug/mL for 1 hour at room temperature followed by incubation with the Anti-Rabbit IgG VisUCyte (TM) HRP Polymer Antibody (Catalog # VC002). Tissue was stained using DAB (brown) and counterstained with hematoxylin (blue). Specific staining was localized to the nuclei.

Simple Western: HIF-1 alpha Antibody (2443C) [NBP2-75978] - Lane view shows a specific band for HIF-1 alpha in HepG2 +/- DFO cell lysate using 20 ug/mL. Electropherogram image of corresponding Simple Western lane view at WES molecular weight of 116 KDa. Image from an internal validation.

Simple Western: HIF-1 alpha Antibody (2443C) [NBP2-75978] - Simple Western lane view shows lysates of MCF +/- DMOG, BioSpherix MSCs in hypoxic conditions, and BioSpherix MSCs in normoxic conditions loaded at 0.5 mg/ml. A band was detected for HIF-1 alpha at approximately 116 kDa (as indicated) using NBP2-75978 (1:100 dilution) followed by Anti-Rabbit Secondary Antibody (042-206, ProteinSimple). This experiment was conducted under standard assay conditions, and using the 12-230 kDa separation module (SW-W004). Non-specific interaction with the 230 kDa Simple Western standard may be seen with this antibody. Image from an internal validation.

Reactivity:

Hu

Species Glossary

Reactivity: Hu

Applications:

, , , ,

Applications: , , , ,

Host:

Rabbit Monoclonal

Host: Rabbit Monoclonal

Sizes:

Sample Size Available

Sizes: Sample Size Available

Conjugate:

Unconjugated

Available Conjugates:

Validated by:

Validated by:

Conjugate	Catalog #	Availability	Size	Price
Alexa Fluor 350	NBP2-75978AF350
Alexa Fluor 405	NBP2-75978AF405
Alexa Fluor 488	NBP2-75978AF488
Alexa Fluor 532	NBP2-75978AF532
Alexa Fluor 594	NBP2-75978AF594
Alexa Fluor 647	NBP2-75978AF647
Alexa Fluor 700	NBP2-75978AF700
Alexa Fluor 750	NBP2-75978AF750
Allophycocyanin	NBP2-75978APC
Biotin	NBP2-75978B
CoraFluor 1	NBP2-75978CL1
DyLight 350	NBP2-75978UV
DyLight 405	NBP2-75978V
DyLight 488	NBP2-75978G
DyLight 550	NBP2-75978R
DyLight 594	NBP2-75978DL594
DyLight 650	NBP2-75978C
DyLight 680	NBP2-75978FR
DyLight 755	NBP2-75978IR
FITC	NBP2-75978F
HRP	NBP2-75978H
Janelia Fluor 525	NBP2-75978JF525
Janelia Fluor 549	NBP2-75978JF549
Janelia Fluor 646	NBP2-75978JF646
Janelia Fluor 669	NBP2-75978JF669
PE	NBP2-75978PE
PerCP	NBP2-75978PCP
mFluor Violet 450 SE	NBP2-75978MFV450
mFluor Violet 500 SE	NBP2-75978MFV500
mFluor Violet 610 SE	NBP2-75978MFV610

Available Formulations

Formulation	Catalog #	Availability	Price
BSA Free	NBP2-75978SS
Azide and BSA Free	NBP2-80765

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NB100-132

HIF-2 alpha/EPAS1 Antibody (ep190b)

(9 Reviews)

(192 Publications)

0.1 ml

NB100-132

Immunohistochemistry-Paraffin: HIF-2 alpha/EPAS1 Antibody (ep190b) [NB100-132] - Analysis of HIF-2 in human cardiac myocytes using HIF-2 alpha/EPAS1 Antibody (ep190b).

Flow Cytometry: HIF-2 alpha/EPAS1 Antibody (ep190b) [NB100-132] - HIF-2 alpha antibody was tested at 1:400 in HepG2 cells using an Alexa Fluor 488 secondary (shown in purple). M1 is defined by unstained cells.

Western Blot: HIF-2 alpha/EPAS1 Antibody (ep190b) [NB100-132] - Mouse aortic endothelial cells treated (1%) or not treated (20.9%) in hypoxia for 3 hrs. Cells where also transfected with a specific siRNA against (siHIF-2) or a control siRNA (-). Western blot image submitted by a verified customer review.

Immunohistochemistry-Paraffin: HIF-2 alpha/EPAS1 Antibody (ep190b) [NB100-132] - Renal tubule specific models of Vhl deletion. Histological images of representative renal sections from 12 month old control, Pax8-CreERT2/Vhldelta/delta and Slc22a6-CreERT2/Vhldelta/delta mice (stains and antibodies as indicated, arrowheads indicate abnormal vascularization). Scale bars, 100 um. Image collected and cropped by CiteAb from the following publication (//dx.plos.org/10.1371/journal.pone.0148055), licensed under a CC-BY license.

<b>Biological Strategies Validation. </b>Western Blot: HIF-2 alpha/EPAS1 Antibody (ep190b) [NB100-132] - Analysis of HepG2 without Cobalt (II) Chloride (1), HepG2 with Cobalt (II) Chloride (2), HepG2 normoxic (3), HepG2 hypoxic (4), HepG2 without Cobalt (II) Chloride (5), HepG2 with Cobalt (II) Chloride (6), HepG2 normoxic (7), and HepG2 hypoxic (8) using this antibody (NB100-132) at 1 - 2 ug/mL.

<b>Biological Strategies Validation. </b>Western Blot: HIF-2 alpha/EPAS1 Antibody (ep190b) [NB100-132] - [HRP] [NB100-132H] - Analysis of HIF-2 alpha stabilization over time in HOS cells following exposure to hypoxia. Image using the HRP form of this antibody (NB100-132H). Image collected and cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/23785417/) licensed under a CC-BY license.

<b>Biological Strategies Validation. </b>Western Blot: HIF-2 alpha/EPAS1 Antibody (ep190b) [NB100-132] - Western blot analysis and quantification of HIF-2 alpha expression in the cortex 4.5 hours after lipopolysaccharide (LPS) administration for all LPS groups and the control group (SHAM, white bar). With the exception of the vagotomy group (VGX LPS, gray bar), no significant differences to the SHAM group were found in the LPS-treated and sham-operated (SHAM+LPS, light gray bar) or vagus nerve-stimulated groups (VGX LPS+STIM, black bar). The significant increase in the VGX LPS (gray bar) group is an indicator of a hypoxic condition; * P<0.05 compared to SHAM; n=6 rats each. Data are given as the mean+/-SEM. Image collected and cropped by CiteAb from the following publication (//jneuroinflammation.biomedcentral.com/articles/10.1186/1742-2094-9-183), licensed under a CC-BY license.

<b>Biological Strategies Validation. </b>Western Blot: HIF-2 alpha/EPAS1 Antibody (ep190b) [NB100-132] - 4-OOHCPA exposure induced HIF-2 alpha/EPAS1. A: Western blot analysis of HIF-2 alpha/EPAS1 protein (118KD) and actin (43KD) in limbs at 10 min, 1, 3, 6, and 24 h after treatment with 4-OOHCPA at 0.3 ug/mL (L) 1.0 ug/mL (M) or 3.0 ug/mL (H). P represents the positive control. Image collected and cropped by CiteAb from the following publication (//dx.plos.org/10.1371/journal.pone.0051937), licensed under a CC-BY license.

Immunohistochemistry: HIF-2 alpha/EPAS1 Antibody (ep190b) [NB100-132] - Analysis of HIF-2 alpha in human endometrium using. Donkey anti-mouse Alexa Fluor 488 secondary antibody was used. IHC image submitted by a verified customer review.

<b>Biological Strategies Validation and Genetic Strategies Validation.</b>Western Blot: HIF-2 alpha/EPAS1 Antibody (ep190b) [NB100-132] - Functional role of HIF2A in the transcriptional regulation of amphiregulin (AREG) in human cardiac myocytes. Immunoblot for HIF1A or HIF2A from shRNA-transfected normoxic or hypoxic HCM. Beta-Actin (ACTb) served as a loading control. Image collected and cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/29483579/) licensed under a CC-BY license.

Immunohistochemistry-Paraffin: HIF-2 alpha/EPAS1 Antibody (ep190b) [NB100-132] - HIF-1 alpha & HIF-2 alpha IHC signals in hypoxic areas of transgenic mouse mammary tumours.A, C Hypoxyprobe (Hyp.pr.) allows visualisation of hypoxic tumour areas. B. Hypoxic peri-necrotic HIF-1 alpha -positive cells display nuclear staining. D. HIF-2 alpha -positive cells show cytoplasmic staining with or without appreciable nuclear staining. * necrosis. E, F. IHC staining for HIF-1 alpha on MCF-7 breast cancer cells grown under control (E) & hypoxic (F) conditions respectively. G, H. Control (G) & hypoxic (H) MCF-7 cells IHC stained for HIF-2 alpha . 20x obj. Insets show magnification of the boxed area. Size bars 50 μm. Image collected & cropped by CiteAb from the following publication (//dx.plos.org/10.1371/journal.pone.0125771), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Immunohistochemistry: HIF-2 alpha/EPAS1 Antibody (ep190b) [NB100-132] - HIF-2 alpha expression in the involuting mammary gland.Inserts are enlargements of the indicated areas. Size bars: 50 μm, 40x obj was used in all micrographs. A. In the early involuting gland, the morphology resembles the lactating gland & the basement membrane is evident at this stage. B-D. As tissue remodelling proceeds during involution, the collagen layer becomes unstructured. E-H. HIF-2 alpha -positive cells were detected at all studied stages of involution. I-L. Macrophage infiltration (F4/80 positive) was first evident at the fifth day of involution (J) & increased with time (K, L). Image collected & cropped by CiteAb from the following publication (//dx.plos.org/10.1371/journal.pone.0125771), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: HIF-2 alpha/EPAS1 Antibody (ep190b) [NB100-132] - Oxygen concentration-dependence of dioxygenase inhibition.A. HIF1 alpha , HIF2 alpha , & H3K9me2 abundance by western blotting in shRNA1 & shRNA2 cells incubated in 21%, 10%, or 2% oxygen for 72 h. Actin & total H3 serve as loading controls. B. HIF1 alpha , HIF2 alpha , & H3K9me2 abundance by western blotting in SDHC knockout iMEFs incubated in 21%, 10%, or 2% oxygen for 72 h. iMEFs were treated with 1 μM TAM for 7 d prior to analysis. (C-D) Rescue of succinate inhibition of JMHD & PHD inhibition using 0.25 mM octyl-alpha -ketoglutarate (octyl-alpha -KG) in SDHB knockdown HEK293 cells & SDHC knockout mouse iMEFs. Image collected & cropped by CiteAb from the following publication (//dx.plos.org/10.1371/journal.pone.0127471), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Immunohistochemistry-Paraffin: HIF-2 alpha/EPAS1 Antibody (ep190b) [NB100-132] - Lactating mammary gland.Smaller panels display enlargements of the indicated areas. Size bars: 50 μm, 40x obj was used for all micrographs. A. Collagen I IHC allows visualisation of the basement membrane surrounding the dilated ducts. B. HIF-1 alpha was not detected in the epithelial cells of the lactating gland (compare with Fig 1). C. Macrophage infiltration was sparse in the lactating mammary gland as judged by F4/80 IHC. D. A subset of cuboidal luminal epithelial cells was distinctively positive for HIF-2 alpha . E. The percentage of HIF-2 alpha -positive out of total luminal epithelial cells was counted in sections from three mice. Image collected & cropped by CiteAb from the following publication (//dx.plos.org/10.1371/journal.pone.0125771), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Immunohistochemistry: HIF-2 alpha/EPAS1 Antibody (ep190b) [NB100-132] - Mainly CK14-positive, but not CK8-positive, mammary epithelial cells are HIF-2 alpha positive at day 14 post weaning.Left panels 20x & right 40x objective. Size bars 50 μm. a, A, b & B. Double IHC for HIF-2 alpha (green) & CK8 (a, A) & CK14 (b, B), respectively (red), reveal that few (if any) CK8-positive luminal cells are HIF-2 alpha positive. Numerous CK14-expressing cells, which include basal & stem/progenitor cells, were positive for HIF-2 alpha . c, C. F4/80 IHC was performed on an adjacent tissue section detect macrophages. Image collected & cropped by CiteAb from the following publication (//dx.plos.org/10.1371/journal.pone.0125771), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Immunohistochemistry: HIF-2 alpha/EPAS1 Antibody (ep190b) [NB100-132] - Renal tubule specific models of Vhl deletion.(A) PCR analysis of recombination at the Vhl locus in the kidneys of mice with combinations of Pax8-CreERT2, Slc22a6-CreERT2 & the Vhl floxed (fl) & wild-type (+) alleles. The positions of the bands representing the Vhl floxed, wild type (Wt) & recombined ( delta ) alleles are indicated. (B) Histological images of representative renal sections from 12 month old control, Pax8-CreERT2/Vhl delta / delta & Slc22a6-CreERT2/Vhl delta / delta mice (stains & antibodies as indicated, arrowheads indicate abnormal vascularisation). Scale bars, 100μm. Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/26866916), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Immunohistochemistry-Paraffin: HIF-2 alpha/EPAS1 Antibody (ep190b) [NB100-132] - HIF1 alpha & H3K9me2 accumulation & 5-hydroxy-methyl-2’-deoxycytidine (5hmdC) depletion in PGL specimens compared to controls.Normal ganglia 1 (NG1), normal ganglia 2 (NG2) & IDH-mutant (IDH). Sporadic PGL (Spo. PGL). A. HIF1 alpha staining. B. HIF2 alpha staining. C. H3K9me2 staining. Arrows indicate H3K9me2 staining in nuclei of neurons or chief cells. D. 5hmdC staining. Arrows indicate 5hmdC staining in the nuclei of neurons & chief cells. Image collected & cropped by CiteAb from the following publication (//dx.plos.org/10.1371/journal.pone.0127471), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Immunohistochemistry: HIF-2 alpha/EPAS1 Antibody (ep190b) [NB100-132] - Localization of HIF2A immunoreactivity in limbs.HIF-2A reactivity was detected in control limbs at 1 & 3 h in the apical ectodermal ridge, interdigital area (I.R.) & developing cartilaginous anlagen (Digits). 4-OOHCPA exposure increased HIF2A immunoreactivity in the apical ectodermal ridge & interdigital regions (I.R.) at 3 h. No differences were observed between control & drug-treated limbs after 6 h or 24 h of culture. Four separate replicates were done. Image collected & cropped by CiteAb from the following publication (//dx.plos.org/10.1371/journal.pone.0051937), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: HIF-2 alpha/EPAS1 Antibody (ep190b) [NB100-132] - Hypoxic repression of ER-alpha is dependent on HIF-1 alpha . a Representative western blots of HIF-1 alpha , HIF-2 alpha & beta -actin protein in ten ER-positive cell lines grown at normoxia or hypoxia (1% O2, 24 h). b qPCR analysis of HIF1A mRNA levels in MCF7, BT474, T47D & ZR75B transfected with shScramble or shHIF1A. Relative HIF1A mRNA levels normalized to TBP. (Change in HIF1A levels: *p < 0.0001 MCF7, *p = 0.012 BT474, *p < 0.0001 T47D, *p = 0.0046 ZR75B). c Representative western blots of HIF-1 alpha , ER-alpha & beta -actin protein from MCF7, BT474, T47D & ZR75B with either shScramble or shHIF1A at normoxia & hypoxia (1% O2, 24 h). beta -actin is used as a loading control. d Representative western blot of HIF-1 alpha , ER-alpha & beta -actin protein from MCF7, BT474, T47D & ZR75B with or without the transfection of stabilized HIF-1 alpha (HIF-1 alpha ODD) Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/28320353), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Immunohistochemistry: HIF-2 alpha/EPAS1 Antibody (ep190b) [NB100-132] - HIF-1 alpha & HIF-2 alpha expression in the virgin mammary gland.A. Virgin mammary glands (30 & 70 days old) showed no conspicuous basal membrane, as visualised by collagen IV IHC (a, b). There was also no detectable expression of HIF-2 alpha in the epithelial cells (c, d). Macrophages (F4/80 positive) were few. In panel c, a single HIF-2 alpha -positive cell was detected, & the adjacent F4/80 IHC section (e) suggested that this cell is a macrophage. B. Expression of HIF-1 alpha in mammary epithelium in the 70-day-old virgin mouse. Top panel, orientation slide with haematoxylin (HTX) staining, 20x obj. *lymph node. Panels b, d, f. Cross-section of a developing duct close to the invading tip at a stage where the lumen is not yet evacuated, 40x obj. Panels c, e, g. Cross-section of a less mature part of a duct, 40x obj. CK14-expressing cells (marker of basal mammary epithelial cells) can be seen in more than one cell layer (panels b & c, arrow-head). At this stage, the lumen is evacuated, but there is still more than one layer of epithelial cells. HIF-1 alpha IHC on the adjacent sections (panels d, e) showing nuclear expression in non-basal epithelial cells (highlighted by red arrows). Basal (CK14 positive) epithelial cells did not express HIF-1 alpha (black arrows). Mammary epithelial expression of HIF-2 alpha was not detected at these developmental stages. Size bars: 50 μm. Image collected & cropped by CiteAb from the following publication (//dx.plos.org/10.1371/journal.pone.0125771), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: HIF-2 alpha/EPAS1 Antibody (ep190b) [NB100-132] - HIF2 alpha inhibitor 76 suppressed CoCl2-induced immature phenotypic characteristics of BCSCs(A) The inhibitory effect of small molecule HIF2 alpha inhibitor 76 for 24h (10 μM for 4T1 cells; 25 μM for Hs578T cells) on CoCl2-induced expression of HIF2 alpha was assessed in both 4T1 & Hs578T cells by western blot analysis. (B) HIF2 alpha inhibitor 76 inhibited primary (with HIF2 alpha inhibitor 76 daily treatment) & second sphere formation (without additional HIF2 alpha inhibitor 76 treatment) in both 4T1 & Hs578T cells. The sizes of spheres greater than 100 μm were enumerated, with a representative image of a tumor-sphere shown. The data represents an average of three independent experiments. (C) Treatment of 4T1 & Hs578T cells with HIF2 alpha inhibitor 76 for 24 h led to a decrease in the percentage of CD44+/CD24−-positive cells as a proportion of total cancer cells. (D) 4T1 & Hs578T cells treated with CoCl2 (100 μM) for 24h & HIF2 alpha inhibitor 76 for 24h (10 μM for 4T1 cells; 25 μM for Hs578T cells) either alone or together were evaluated for the expression levels of stem cell markers c-Myc, Klf4, Oct4, & Nanog by Real-time PCR. Abbreviations: TSFE, Tumor sphere-forming efficiency. beta -actin was used as the internal control. The results represent the mean ± SD from three independent experiments. Image collected & cropped by CiteAb from the following publication (//www.oncotarget.com/lookup/doi/10.18632/oncotarget.9846), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: HIF-2 alpha/EPAS1 Antibody (ep190b) [NB100-132] - Phd2 deletion leads to activation of the Akt–mTOR pathway. a RPPA analysis of tumors with homozygous deletion of Phd2. Tumor tissues from Tyr::CreER; BRafV600E; Phd2−/− or Tyr::CreER; BRafV600E mice were processed & analyzed by RPPA assays. The analyses identified proteins that were significantly changed in mouse melanomas compared to nevi. b Activation of Akt–mTOR pathway after phd2 deletion. Tumor tissues were processed & western blots showed stabilization of HIF-1 alpha & HIF-2 alpha proteins after Phd2 depletion. Increased phosphorylation of Akt, 4EBP1 & S6K was observed in tumors from Tyr::CreER; BRafV600E; Phd2−/− compared with those of Tyr::CreER; BRafV600E mice. c Re-expression of Phd2 inhibits the Akt–mTOR pathway. A BRafV600E; Phd2−/− mouse melanoma cell line was established from melanomas in Tyr::CreER; BRafCA; Phd2lox/lox mice. Phd2 was ectopically reintroduced in these tumor cells. Western blot analysis showed that degradation of HIF-1 alpha & HIF-2 alpha proteins with decreased expression of VEGFR2 decreased phosphorylation of Akt, 4EBP1 & S6K. d Pharmacological inhibition (FM19G11) of HIF pathway in BRafV600E; Phd2−/− melanoma cells. A similar but more pronounced inhibition of the Akt–mTOR pathway was observed using the HIF inhibitor. beta -Actin was used as a loading control. Results are representative of three independent experiments Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/30575721), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: HIF-2 alpha/EPAS1 Antibody (ep190b) [NB100-132] - BAY 87-2243 inhibits hypoxia-inducible factor (HIF-1 alpha ) & HIF-2 alpha protein accumulation in hypoxic H460 cells H460 under hypoxia but has no effect on HIF-1 alpha protein levels induced by hypoxia mimetics & has no effect on prolyl hydroxylase 2 (PHD2) activity. (A, B) H460 cells were cultured for 16 h under normoxia or hypoxia (1% pO2) in the absence or presence of various concentrations of BAY 87-2243. HIF-1 alpha (A) & HIF-2 alpha (B) protein levels were assessed by Western Blot in whole cell extracts. beta -actin was used as a loading control. (C) H460 cells were cultured for 16 h under normoxia with the PHDs desferrioxamine (DFO) & CoCl2 plus/minus BAY 87-2243 before the HIF1 alpha protein levels in cellular extracts were quantified by Western Blot. beta -actin was used to as a loading control. (D) Effect of BAY 87-2243 on the recombinant PHD2-mediated hydroxylation of HIF-1 alpha peptide over time was measured in a biochemical assay. Hydoxylated peptide was quantified after incubation with purified VBC complex labeled with europium using fluorescence as a readout. The known PHD inhibitor N-oxalylglycine served as a positive control. Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/24403227), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: HIF-2 alpha/EPAS1 Antibody (ep190b) [NB100-132] - Cytosol-restricted Acss2 is enzymatically active.(A) Acetylation of ectopic HA-tagged HIF-2 alpha detected by immunoblotting (IB) with anti-HA or anti- acetylated lysine antibodies following immunoprecipitation (IP) with anti-HA antibody in stably transformed HT1080 cells with knockdown of endogenous Acss2 & rescue with ectopic wild-type (WT) or cytosol-restricted mutant (CYT) Acss2 without or with an SV40 nuclear localization signal fused to the amino terminus. Studies were performed under hypoxia, low glucose, or acetate exposure for the indicated periods. (B) Acetate-dependent lipid synthesis measured by 14C-acetate incorporation in HT1080 stably-transformed cells producing control or Acss2 shRNA downstream of a luciferase cDNA cassette & expressing ectopic control, WT, CYT, SV40-WT, or SV40-CYT Acss2. Cells were incubated under (A) control, (B) hypoxic, or (C) low glucose conditions for 48 hr with labeling performed during the last 24 hr. Comparison of samples within a given condition was made by one-way ANOVA followed by Dunnett’s multiple comparisons test using control shRNA knockdown/control rescue as reference with decreased samples noted (*, P<0.05). All values are means with SD. Image collected & cropped by CiteAb from the following publication (//dx.plos.org/10.1371/journal.pone.0190241), licensed under a CC0-1.0 license. Not internally tested by Novus Biologicals.

Western Blot: HIF-2 alpha/EPAS1 Antibody (ep190b) [NB100-132] - In vitro effects of diclofenac on proliferation & MYC expression in the human melanoma cell line MelIm.The human melanoma cell line MelIm was incubated with different concentrations of diclofenac (A), aspirin (ASA, B), & NS-398 (C), respectively, & proliferation was determined after 24 h. Results represent the mean +/− standard deviation of 12 (diclofenac) & 3 (ASA, NS-398) independent experiments, respectively. (D) MelIm were incubated for 24 h with or without diclofenac. Apoptotic cells were stained with Annexin-V-FITC/ 7-AAD & analyzed by flow cytometry. Results represent the mean +/− standard deviation of 3 independent experiments. (E-G) MYC, STAT3, HIF1a & HIF2a protein expression were determined in cell lysates of MelIm incubated for 2 or 24 h with or without diclofenac (E,F) or ASA (G). The effect of diclofenac on MYC promoter activity was determined by transient transfection of a 2632-bp MYC promoter fragment (H). MelIm were transfected in 6-well-plates & diclofenac was added after 5 h. Luciferase activity was determined 24 h after transfection. Results represent the mean +/− standard deviation of 3 independent experiments. Image collected & cropped by CiteAb from the following publication (//dx.plos.org/10.1371/journal.pone.0066987), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Immunohistochemistry: HIF-2 alpha/EPAS1 Antibody (ep190b) [NB100-132] - In-vivo lentiviral delivery of Cre-recombinase to renal tubular epithelium results in recombination of target genes.(a) Diagram of pCCIE lentiviral construct. Cre, Cre-recombinase; IRES, internal ribosome entry site; GFP, Green fluorescent protein. (b) Anti-VHL, HIF1a & GAPDH immunoblots of renal cortical protein lysates from Vhlwt/wt & Vhlfl/fl mice intrarenally injected with CCIE. Samples were collected 12 months post infection with each column representing an individual mouse. Blots were cropped to improve clarity, full-length blots are presented in Supplementary Fig. S3a. (c) Histological images of renal sections from Vhlwt/wt & Vhlfl/fl mice intrarenally injected with CCIE at 12 months post injection (stains & antibodies as indicated). Scale bars, 100 μm. Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/26046460), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Immunohistochemistry-Paraffin: HIF-2 alpha/EPAS1 Antibody (ep190b) [NB100-132] - HIF-1 alpha & HIF-2 alpha expression in the virgin mammary gland.A. Virgin mammary glands (30 & 70 days old) showed no conspicuous basal membrane, as visualised by collagen IV IHC (a, b). There was also no detectable expression of HIF-2 alpha in the epithelial cells (c, d). Macrophages (F4/80 positive) were few. In panel c, a single HIF-2 alpha -positive cell was detected, & the adjacent F4/80 IHC section (e) suggested that this cell is a macrophage. B. Expression of HIF-1 alpha in mammary epithelium in the 70-day-old virgin mouse. Top panel, orientation slide with haematoxylin (HTX) staining, 20x obj. *lymph node. Panels b, d, f. Cross-section of a developing duct close to the invading tip at a stage where the lumen is not yet evacuated, 40x obj. Panels c, e, g. Cross-section of a less mature part of a duct, 40x obj. CK14-expressing cells (marker of basal mammary epithelial cells) can be seen in more than one cell layer (panels b & c, arrow-head). At this stage, the lumen is evacuated, but there is still more than one layer of epithelial cells. HIF-1 alpha IHC on the adjacent sections (panels d, e) showing nuclear expression in non-basal epithelial cells (highlighted by red arrows). Basal (CK14 positive) epithelial cells did not express HIF-1 alpha (black arrows). Mammary epithelial expression of HIF-2 alpha was not detected at these developmental stages. Size bars: 50 μm. Image collected & cropped by CiteAb from the following publication (//dx.plos.org/10.1371/journal.pone.0125771), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Reactivity:

Hu, Mu, Rt, Bv, Ha

Species Glossary

Reactivity: Hu, Mu, Rt, Bv, Ha

Applications:

, , , , , , , , , , ,

Applications: , , , , , , , , , , ,

Host:

Mouse Monoclonal

Host: Mouse Monoclonal

Sizes:

Sample Size Available

Sizes: Sample Size Available

Conjugate:

Unconjugated

Available Conjugates:

Validated by:

Validated by:

Conjugate	Catalog #	Availability	Size	Price
Alexa Fluor 350	NB100-132AF350
Alexa Fluor 405	NB100-132AF405
Alexa Fluor 488	NB100-132AF488
Alexa Fluor 532	NB100-132AF532
Alexa Fluor 594	NB100-132AF594
Alexa Fluor 647	NB100-132AF647
Alexa Fluor 700	NB100-132AF700
Alexa Fluor 750	NB100-132AF750
Allophycocyanin	NB100-132APC
Biotin	NB100-132B
CoraFluor 1	NB100-132CL1
DyLight 350	NB100-132UV
DyLight 405	NB100-132V
DyLight 488	NB100-132G
DyLight 550	NB100-132R
DyLight 594	NB100-132DL594
DyLight 650	NB100-132C
DyLight 680	NB100-132FR
DyLight 755	NB100-132IR
FITC	NB100-132F
HRP	NB100-132H
Janelia Fluor 525	NB100-132JF525
Janelia Fluor 669	NB100-132JF669
PE	NB100-132PE
PerCP	NB100-132PCP
mFluor Violet 450 SE	NB100-132MFV450
mFluor Violet 500 SE	NB100-132MFV500
mFluor Violet 610 SE	NB100-132MFV610

Available Formulations

Formulation	Catalog #	Availability	Price
Azide and BSA Free	NBP2-80766

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NB100-123

HIF-1 alpha Antibody (H1alpha67)

(9 Reviews)

(162 Publications)

0.1 ml

NB100-123

<b>Biological Strategies Validation. </b>Western Blot: HIF-1 alpha Antibody (H1alpha67) [NB100-123] - MLN4924 induces accumulation of HIF1a in a time dependent manner. Cells were treated with 0.1uM MLN4924 for indicated time periods, followed by IB with indicated antibodies. Image collected and cropped by CiteAb from the following publication (//www.nature.com/articles/cddis2012125) licensed under a CC-BY license.

Immunohistochemistry: HIF-1 alpha Antibody (H1alpha67) [NB100-123] - Analysis of HIF-1 alpha in human lung tissue. Image courtesy of product review by Aneta Gandjeva.

Immunoprecipitation: HIF-1 alpha Antibody (H1alpha67) [NB100-123] - MG132 attenuates the inhibitory effect of interleukin-1beta on HIF-1/AM axis. U87MG cells were incubated for 2 h in hypoxia (1% O2) with or without interleukin-1beta (10 ng/ml). The proteasome inhibitor MG132 was added 30 min in advance to prevent ubiquitinated HIF-1alpha from proteasomal degradation. HIF-1alpha was precipitated by anti-HIF-1alpha antibody. Ubiquitin was detected by immunoblotting. Non-specific mouse IgG was used as a negative control. The results are representative for three independent experiments Image collected and cropped by CiteAb from the following publication (//www.nature.com/articles/cddis2013562) licensed under a CC-BY license.

<b>Biological Strategies Validation. </b>Western Blot: HIF-1 alpha Antibody (H1alpha67) [NB100-123] - Analysis using the HRP conjugate of NB100-123. Detection of HIF-1 alpha in cobalt chloride treated/untreated COS-7 nuclear extracts using NB100-123.

$<b>Biological Strategies Validation. </b>Western Blot: HIF-1 alpha Antibody (H1alpha67) [NB100-123] - Analysis using the HRP conjugate of NB100-123. On day 1, MEF cells (+/+,-/-), were grown on 15cm dish (2x10 to the 6th cells). On day 2, cells were exposed to hypoxia for 4hrs. Cells were washed with ice cold PBS twice and whole cell protein was extracted extracted with RIPA buffer fortified with protease. Upon quantification, 100ug of protein was fractionated on 7% polyacralymide gel. Gel was transferred overnight onto nitrocellulose membrane. The membrane was probed with HIF-1 alpha monoclonal antibody at a 1:500 dilution (NB100-123). The secondary antibody was conjugated with HRP and was used at a 1:2500 dilution. Photo courtesy of Dr. Gregg Semenza, Johns Hopkins University.$

<b>Biological Strategies Validation. </b>Immunocytochemistry/Immunofluorescence: HIF-1 alpha Antibody (H1alpha67) [NB100-123] - Dapagliflozin protects against metabolic switch by inhibition of HIF-1 alpha. Representative images of PTCs incubated with 10 umol/l of molidustat for 6 h with immunofluorescent staining for HIF-1 alpha. Scale bar = 10 um. Image collected and cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/33187129/) licensed under a CC-BY license.

Immunohistochemistry-Paraffin: HIF-1 alpha Antibody (H1alpha67) [NB100-123] - Immunohistochemical detection of HIF-1alpha protein in human tissues. The nature of the tissue is indicated on top of each figure. Original magnifications are as follows: A, x400; B, x400; C, x100; D, x100; E, x100; F, x100; G, x40; H, x40. Image collected and cropped by CiteAb from the following publication (//bmcgenet.biomedcentral.com/articles/10.1186/1471-2156-5-27), licensed under a CC-BY license.

Immunocytochemistry/Immunofluorescence: HIF-1 alpha Antibody (H1alpha67) [NB100-123] - Staining in pig endothelial cells under hypoxia condition using NB100-123. Image from verified customer review.

Immunohistochemistry-Paraffin: HIF-1 alpha Antibody (H1alpha67) [NB100-123] - Staining on pig tissue (brown) using NB100-123. Image from verified customer review.

Immunohistochemistry: HIF-1 alpha Antibody (H1alpha67) [NB100-123] - Staining of biotin conjugated HIF-1 alpha (NB100-123B) in human kidney.

Immunoprecipitation: HIF-1 alpha Antibody (H1alpha67) [NB100-123] - Immunoprecipitation of HIF-1alpha using NB100-123 in COS7 CoCl2 treated lysate. Heavy and light chains are also detected. Image using the HRP form of this antibody.

Immunohistological and western blot analyses of HIF-1 alpha .(A) PAB liver stained by HIF-1 alpha (original magnification, ×20). (B) Sham-operated control liver stained by HIF-1 alpha (original magnification, ×20). (C) Enlarged view of the boxed area in the Image (A) (original magnification, ×100). (D) Western blot analysis of HIF-1 alpha (mean ± SEM), * P<0.05. HIF-1 alpha : hypoxia-inducible factor-1 alpha ; PAB: pulmonary artery banding.

<b>Biological Strategies Validation. </b>Immunocytochemistry/Immunofluorescence: Mouse Monoclonal HIF-1 alpha Antibody (H1alpha67) [NB100-123] - Images of HIF-1 alpha localization (red) in CTR and CoCl2-treated oAECs after 24 h of culture. Image from a verified customer review.

Western Blot: Mouse Monoclonal HIF-1 alpha Antibody (H1alpha67) [NB100-123] - Image of HIF-1 alpha activation in AECs control cells (CTR) and after 24h of 10 and 50µM of CoCl2-treatment; HIF-1 alpha time course at shorter CoCl2 incubation times. Image from a verified customer review.

Immunohistochemistry: HIF-1 alpha Antibody (H1alpha67) [NB100-123] - Immunohistochemical detection of HIF-1 alpha protein in human tissues. The nature of the tissue is indicated on top of each figure. Original magnifications are as follows: A, ×400; B, ×400; C, ×100; D, ×100; E, ×100; F, ×100; G, ×40; H, ×40. Image collected & cropped by CiteAb from the following publication (//bmcgenet.biomedcentral.com/articles/10.1186/1471-2156-5-27), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: HIF-1 alpha Antibody (H1alpha67) [NB100-123] - Immunohistological & western blot analyses of HIF-1 alpha .(A) PAB liver stained by HIF-1 alpha (original magnification, ×20). (B) Sham-operated control liver stained by HIF-1 alpha (original magnification, ×20). (C) Enlarged view of the boxed area in the Image (A) (original magnification, ×100). (D) Western blot analysis of HIF-1 alpha (mean ± SEM), * P<0.05. HIF-1 alpha : hypoxia-inducible factor-1 alpha ; PAB: pulmonary artery banding. Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/26863419), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Immunocytochemistry/ Immunofluorescence: HIF-1 alpha Antibody (H1alpha67) [NB100-123] - Analysis of CBF treatment in HCT116 implanted mouse models & tumour tissue samples. (a) Tumour size versus CBF treatment. The lowest tumour growth rate was found in i.p. group during CBF treatment. (b) Analysis of HIF-1 alpha mRNA level in tumour tissues. There was a significant upregulated HIF-1 alpha level in i.p. group. Results are means with standard errors from 4 replicates. (c) Inhibition of HIF-1 alpha nuclear translocation in the i.t. sample. CBF strongly inhibited HIF-1 alpha nuclear accumulation in the tumour of xenografts implanted with HCT116 cells. Scale bars equal 10 μm. White arrows: HIF-1 alpha in the cytosol. Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/23818933), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Immunohistochemistry: HIF-1 alpha Antibody (H1alpha67) [NB100-123] - CycT alleviates tumor hypoxia. The effects of CycT on the levels of exogenous hypoxia-marker pimonidazole labeling (A), the levels of hypoxia-inducible CA9 enzyme (B), & the levels of hypoxia-inducible factor HIF1 alpha (C) in orthotopic tumor xenografts. Scale bar: Montage, 1 mm; 10X, 20 μm. Data are plotted as mean ± SEM. For statistical analysis, the levels in treated tumors were compared to the levels in control tumors with a Welch 2-sample t-test. **p-value < 0.005. IHC images are representative of 3 independent experiments. Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/30723259), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Immunohistochemistry: HIF-1 alpha Antibody (H1alpha67) [NB100-123] - Schematic demonstration of the three HIF1 alpha antibodies used in this study & their epitopes (Figs. 2a & b). Ab1 is a polyclonal antibody raised against N-domain of wild type HIF1 alpha & does not recognize HIF1 alpha 1.2 due to it's different N-terminal part (Fig. 2a). Ab2 & Ab3 are monoclonal & polyclonal antibodies, respectively, with epitopes in the common parts of HIF1 alpha & HIF1 alpha 1.2 (Fig.2a & b). Immunohistochemical analysis performed on thin adjacent sections of NE-differentiated prostate cancer using the HIF1 alpha antibodies Ab1 (Fig. 2c), Ab 2 (Fig. 2d), Ab 3 (Fig. 2e) & HIF1 beta antibody (Fig. 2f). Immunopositivity was detected for HIF1 alpha with Ab2 & Ab3 while HIF1 alpha Ab1 produced no detectable staining. HIF1 beta was also positive in adjacent section (Fig. 2f). Double staining of chromogranin A & androgen receptor antigens on adjacent sections (Fig. 2g) showed immunopositivity for chromogranin A (Fast red). Androgen receptor antibody (DAB, brown) produced no staining. Immunostaining of benign prostate tissue with HIF1 alpha Ab3 showed immunopositivity in NE-like cells of benign prostate tissue (Fig. 2h). In addition, HIF1 beta antibody recognized benign NE-like cells in benign prostate hyperplasia (Fig. 2i). Double staining with HIF1 alpha Ab3 & HIF1 beta (Fig. 2j) shows co-localization of the two proteins in NE-like cells of benign prostate hyperplasia (HIF1 alpha Ab3 red stain, HIF1 beta brown stain). Panels a, b, c, d, e, f & g: 40× objective. Panels h, i & j: 60× objective. Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/20663134), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Immunocytochemistry/ Immunofluorescence: HIF-1 alpha Antibody (H1alpha67) [NB100-123] - GAD65/67-positive neurons expressed HIF-1 alpha under hypoxic conditions. A) HIF-1 alpha expression co-localized with GAD65/67-ir in neurons exposed to hypoxia when compared to normoxia (upper panel) or GAD65/67-negative neurons in hypoxia (bottom panel, open arrow). B) Quantification shows the percentage of HIF-1 alpha -expressing GAD65/67-positive neurons after hypoxia in vitro (mean ± SD; from n = 6 cultures). C)In vivo immunostaining illustrates HIF-1 alpha -positive (bottom panel, solid arrow) & HIF-1 alpha -negative (bottom panel, open arrow) in GAD65/67-ir neurons in the ipsilateral region, whereas the contralateral region shows no HIF-1 alpha staining in GAD65/67-ir neurons (see Figure 1 for region selection). Scale bars, 10 μm (A); 20 μm (B). Image collected & cropped by CiteAb from the following publication (//actaneurocomms.biomedcentral.com/articles/10.1186/2051-5960-2-51), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Immunocytochemistry/ Immunofluorescence: HIF-1 alpha Antibody (H1alpha67) [NB100-123] - Levels of GSH in cortical neurons exposed to hypoxia. A) During hypoxia, GSH increased in MAP2-ir neurons with round somata (bottom panel, open arrow) & decreased in neurons with pyramidal-like morphology (upper panel, solid arrow). B) GSH increased in a subset of GAD65/67–ir neurons exposed to hypoxia (bottom panel, open arrow). C) Elevated HIF-1 alpha expression in GAD65/67–ir neurons containing high levels of GSH when exposed to hypoxia (middle panel). BSO treatment decreased HIF-1 alpha expression in GAD65/67-positive neurons during hypoxia. D) Percentages of HIF-1 alpha -expressing GAD65/67-ir neurons expressing a high level of GSH & GAD65/67-ir neurons expressing a low levels of GSH & HIF-1 alpha (mean ± SD; n = 3). The “low” level referred to the level of GSH in normoxic GAD65/67 neurons that was normalized to 1. The “high” level referred to the elevated GSH level in GAD65/67 neurons with a mean of 1.40 ± 0.10. E) Total MCB-GSH intensity in GAD65/67-positive neurons with & without BSO after hypoxia (mean ± SD; 8-10 neurons quantified from each experiment, n = 3 independent cultures). Scale bars, 20 μm. Image collected & cropped by CiteAb from the following publication (//actaneurocomms.biomedcentral.com/articles/10.1186/2051-5960-2-51), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Immunohistochemistry: HIF-1 alpha Antibody (H1alpha67) [NB100-123] - Results of in situ hybridization & immunohistochemistry on thin adjacent section to detect expression of HIF-1 alpha 1.2 mRNA & HIF1 alpha protein in malignant & benign prostate tissue. In situ hybridization (antisense probe, Fig. 3a) & immunostaining with HIF1 alpha Ab2 (Fig. 3c) on thin adjacent sections of NE-differentiated prostate adenocarcinoma showed co-localization of HIF1 alpha 1.2 transcript & HIF-1 alpha protein. Incubation with sense probe did not generate any detectable hybridization signals (Fig. 3b). Both In situ hybridization (Fig. 3d antisense) & HIF-1 alpha Ab2 immunostaining (Fig. 3f) were negative in non-NE-differentiated prostate adenocarcinoma. In situ hybridization with sense probe performed on non-NE-differentiated prostate cancer (Fig. 3e) was negative. In situ hybridizering on benign prostate tissue showed HIF1 alpha 1.2 transcript in NE-like cells of benign prostate tissue (Fig. 3g, ↗). The sense probe on thin adjacent section generated no signals (Fig. 3h,↗). Furthermore, co-localization of HIF1 alpha 1.2 transcript (Fig. 3i,↗,◢,↖) & HIF1 alpha protein, detected with HIF1 alpha Ab3 (Fig. 3j,↗,◢,↖) was also shown in NE-like cells of benign prostate tissue. Panels a, b, c, d, e, f, g, h, i, j: 40× objective. Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/20663134), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: HIF-1 alpha Antibody (H1alpha67) [NB100-123] - HX-induced Sirt1 expression in white matter OPCs requires HIF1 alpha .(a) HIF1 alpha stabilization of Sirt1 transcript expression in OPCs as revealed by representative RT–PCR represents higher level of Sirt1 mRNA in VHL cKO mice. GDPDH mRNA serves as a control. Mean±s.e.m., n=3 brains for each group. (b,c) Representative western blot demonstrates a transient increase of HIF1 alpha expression in HX white matter at P18 with no significant effect at P11 (P=0.7955) & P45 (P=0.7333). Histograms show mean±s.e.m. (d,e) Graphs represent the percentages of Sirt1+Ki67+ & NG2+Ki67+ cells after HX white matter in WT & HIF1 alpha KO mice. Number in parentheses within bar indicates number of samples (n=4 brains per group & per genotype; ****P<0.0001, one-way analysis of variance, Bonferroni post hoc test, mean±s.e.m.). (f,g) Western blot demonstrates no increase (P=0.8231) in Sirt1 & HIF1 alpha expression in white matter of Hif1 alpha KO mice. Actin was used as loading control (mean±s.e.m.; n=3 brains for each experiment, group & genotype). Image collected & cropped by CiteAb from the following publication (//www.nature.com/articles/ncomms13866), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Immunocytochemistry/ Immunofluorescence: HIF-1 alpha Antibody (H1alpha67) [NB100-123] - HIF-1 alpha expression in primary cortical neurons exposed to hypoxia/ischemia. A & A’) Neurons were double-stained for HIF-1 alpha & MAP2 in the presence of 5 & 25 mM glucose with & without hypoxia. HIF-1 alpha expression in the somata was observed in cells with interneuron-like morphology after hypoxia. B) CoCl2 (0.3 mM) induced HIF-1 alpha expression in cells with interneuron-like morphology. C) Quantification represents the increase in HIF-1 alpha –ir staining (mean ± SD; 5-10 neurons quantified from each experiment, n = 3 experiments). D)In vivo brain slice shows a similar pattern of positive HIF-1 alpha -ir in round soma (open arrow) & negative in neurons with pyramidal-like morphology (solid arrow) in the ipsilateral side. *p < 0.05, compared with normoxia (25 mM glucose), #p < 0.05, compared with hypoxia (25 mM glucose). Scale bars, 20 μm (A, A’, B); 10 μm (D). Image collected & cropped by CiteAb from the following publication (//actaneurocomms.biomedcentral.com/articles/10.1186/2051-5960-2-51), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Reactivity:

Hu, Mu, Rt, Po, Av, Bv, Ca, Ma, Pm, Rb, Sh

Species Glossary

Reactivity: Hu, Mu, Rt, Po, Av, Bv, Ca, Ma, Pm, Rb, Sh

Applications:

, , , , , , , , , , ,

Applications: , , , , , , , , , , ,

Host:

Mouse Monoclonal

Host: Mouse Monoclonal

Sizes:

Sample Size Available

Sizes: Sample Size Available

Conjugate:

Unconjugated

Available Conjugates:

Validated by:

Validated by:

Conjugate	Catalog #	Availability	Size	Price
Alexa Fluor 350	NB100-123AF350
Alexa Fluor 405	NB100-123AF405
Alexa Fluor 488	NB100-123AF488
Alexa Fluor 532	NB100-123AF532
Alexa Fluor 594	NB100-123AF594
Alexa Fluor 647	NB100-123AF647
Alexa Fluor 700	NB100-123AF700
Alexa Fluor 750	NB100-123AF750
Allophycocyanin	NB100-123APC
Biotin	NB100-123B
CoraFluor 1	NB100-123CL1
DyLight 350	NB100-123UV
DyLight 405	NB100-123V
DyLight 488	NB100-123G
DyLight 550	NB100-123R
DyLight 594	NB100-123DL594
DyLight 650	NB100-123C
DyLight 680	NB100-123FR
DyLight 755	NB100-123IR
FITC	NB100-123F
HRP	NB100-123H
Janelia Fluor 525	NB100-123JF525
Janelia Fluor 669	NB100-123JF669
PE	NB100-123PE
PerCP	NB100-123PCP
mFluor Violet 450 SE	NB100-123MFV450
mFluor Violet 500 SE	NB100-123MFV500
mFluor Violet 610 SE	NB100-123MFV610

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At	=	A. thaliana	SyHa	=	Golden Syrian Hamster
All	=	All Species	Ha	=	Hamster
An	=	Animal	Hu	=	Human
ArHa	=	Armenian Hamster	I	=	Insect
Av	=	Avian	Ll	=	Llama
Bb	=	Baboon	Ma	=	Mammal
Ba	=	Bacteria	Mk	=	Monkey
Bv	=	Bovine	Mu	=	Mouse
Ca	=	Canine	Multi	=	Multi-species
Ce	=	C. Elegans	NA	=	Non-species specific
Ch	=	Chicken	Or	=	Orangutan
ChHa	=	Chinese Hamster	Pl	=	Plant
Cm	=	Cynomolgus Monkey	Pm	=	Primate
Do	=	Donkey	Po	=	Porcine
Dr	=	Drosophilia	Rb	=	Rabbit
Ec	=	E. Coli	Rt	=	Rat
Eq	=	Equine	RM	=	Rhesus Macaque
Fe	=	Feline	Sh	=	Sheep
Ft	=	Ferret	Vb	=	Vertebrate
Fi	=	Fish	Vi	=	Virus
Fu	=	Fungi	Xp	=	Xenopus
Ge	=	Gerbil	Ye	=	Yeast
GP	=	Guinea Pig	Ze	=	Zebrafish
Gt	=	Goat

Category

Protein/Target

Applications

Reactivity

Citation

Antibody Validation

Reviews

Host

Available Conjugates

Format

Marker

Modification

Clonality

Clone

Gene Symbol

Images Type

Isotype

Kit Type

Lysate Subcellular Fraction

Lysate Type

Preservative

Protein Or Peptide

Research Areas

Has Sample Size

Promotional Item

Species Glossary

HIF-1 alpha Antibody (H1alpha67)

HIF-2 alpha/EPAS1 Antibody - BSA Free

beta-Actin Antibody (AC-15)

HIF-1 alpha Antibody - BSA Free

Survivin Antibody - BSA Free

IRE1 alpha [p Ser724] Antibody - BSA Free

HIF-1 alpha Antibody

HIF-1 alpha Antibody - BSA Free

HIF-1 alpha Antibody (2443B) - BSA Free

HIF-1 alpha Antibody (2443C) - BSA Free

HIF-2 alpha/EPAS1 Antibody (ep190b)

HIF-1 alpha Antibody (H1alpha67)