Product Search for transcription factor 1: Novus Biologicals

NB100-105

HIF-1 alpha Antibody (H1alpha67)

(45 Reviews)

(1038 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 585	NB100-105JF585
Janelia Fluor 635	NB100-105JF635
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)

(822 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 585	NB100-122JF585
Janelia Fluor 635	NB100-122JF635
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

compare

(select up to 3 products from this page to compare)

NB100-479

HIF-1 alpha Antibody - BSA Free

(28 Reviews)

(517 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 585	NB100-479JF585
Janelia Fluor 635	NB100-479JF635
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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NBL1-16837

Transcription factor E3 Overexpression Lysate

0.1 mg

NBL1-16837

Reactivity:

Hu

Species Glossary

Reactivity: Hu

Applications:

Applications:

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NBP3-20547

Transcription factor E3 Antibody (TFE3/6849R)

100 μg

NBP3-20547

IHC analysis of formalin-fixed, paraffin-embedded human clear cell RCC. Negative tissue control using Transcription factor E3 antibody (TFE3/6849R) at 2ug/ml in PBS for 30min RT. HIER: Tris/EDTA, pH9.0, 45min. Secondary: HRP-polymer, 30min. DAB, 5min.

IHC analysis of formalin-fixed, paraffin-embedded human MiT translocation RCC. Nuclear staining using Transcription factor E3 antibody (TFE3/6849R) at 2ug/ml. HIER: Tris/EDTA, pH9.0, 45min. Secondary: HRP-polymer, 30min. DAB, 5min.

Reactivity:

Hu

Species Glossary

Reactivity: Hu

Applications:

Applications:

Host:

Rabbit Monoclonal

Host: Rabbit Monoclonal

Conjugate:

Unconjugated

Available Conjugates:

Conjugate	Catalog #	Availability	Size	Price
Alexa Fluor 350	NBP3-21005AF350
Alexa Fluor 405	NBP3-21005AF405
Alexa Fluor 488	NBP3-21005AF488
Alexa Fluor 532	NBP3-21005AF532
Alexa Fluor 594	NBP3-21005AF594
Alexa Fluor 647	NBP3-21005AF647
Alexa Fluor 700	NBP3-21005AF700
Alexa Fluor 750	NBP3-21005AF750
Allophycocyanin	NBP3-21005APC
Biotin	NBP3-21005B
CoraFluor 1	NBP3-21005CL1
DyLight 350	NBP3-21005UV
DyLight 405	NBP3-21005V
DyLight 488	NBP3-21005G
DyLight 550	NBP3-21005R
DyLight 594	NBP3-21005DL594
DyLight 650	NBP3-21005C
DyLight 680	NBP3-21005FR
DyLight 755	NBP3-21005IR
FITC	NBP3-21005F
HRP	NBP3-21005H
Janelia Fluor 525	NBP3-21005JF525
Janelia Fluor 549	NBP3-21005JF549
Janelia Fluor 585	NBP3-21005JF585
Janelia Fluor 635	NBP3-21005JF635
Janelia Fluor 646	NBP3-21005JF646
Janelia Fluor 669	NBP3-21005JF669
PE	NBP3-21005PE
PerCP	NBP3-21005PCP

Available Formulations

Formulation	Catalog #	Availability	Price
Azide and BSA Free	NBP3-21005

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H00007030-D01P

Transcription factor E3 Antibody

0.1 mg

H00007030-D01P

Reactivity:

Hu, Mu

Species Glossary

Reactivity: Hu, Mu

Applications:

Applications:

Host:

Rabbit Polyclonal

Host: Rabbit Polyclonal

Conjugate:

Unconjugated

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NBP3-09959

Transcription factor E3 Antibody

100 ul

NBP3-09959

Reactivity:

Hu, Mu

Species Glossary

Reactivity: Hu, Mu

Applications:

Applications:

Host:

Rabbit Polyclonal

Host: Rabbit Polyclonal

Conjugate:

Unconjugated

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NBP3-20548

Transcription factor E3 Antibody (TFE3/8663R)

100 μg

NBP3-20548

IHC analysis of formalin-fixed, paraffin-embedded human thyroid. Nuclear staining using Transcription factor E3 antibody (TFE3/8663R) at 2ug/ml. Inset: PBS instead of primary antibody; secondary only negative control.

Reactivity:

Hu

Species Glossary

Reactivity: Hu

Applications:

Applications:

Host:

Rabbit Monoclonal

Host: Rabbit Monoclonal

Conjugate:

Unconjugated

Available Conjugates:

Conjugate	Catalog #	Availability	Size	Price
Alexa Fluor 350	NBP3-21006AF350
Alexa Fluor 405	NBP3-21006AF405
Alexa Fluor 488	NBP3-21006AF488
Alexa Fluor 532	NBP3-21006AF532
Alexa Fluor 594	NBP3-21006AF594
Alexa Fluor 647	NBP3-21006AF647
Alexa Fluor 700	NBP3-21006AF700
Alexa Fluor 750	NBP3-21006AF750
Allophycocyanin	NBP3-21006APC
Biotin	NBP3-21006B
CoraFluor 1	NBP3-21006CL1
DyLight 350	NBP3-21006UV
DyLight 405	NBP3-21006V
DyLight 488	NBP3-21006G
DyLight 550	NBP3-21006R
DyLight 594	NBP3-21006DL594
DyLight 650	NBP3-21006C
DyLight 680	NBP3-21006FR
DyLight 755	NBP3-21006IR
FITC	NBP3-21006F
HRP	NBP3-21006H
Janelia Fluor 525	NBP3-21006JF525
Janelia Fluor 549	NBP3-21006JF549
Janelia Fluor 585	NBP3-21006JF585
Janelia Fluor 635	NBP3-21006JF635
Janelia Fluor 646	NBP3-21006JF646
Janelia Fluor 669	NBP3-21006JF669
PE	NBP3-21006PE
PerCP	NBP3-21006PCP

Available Formulations

Formulation	Catalog #	Availability	Price
Azide and BSA Free	NBP3-21006

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NBP3-21006

Transcription factor E3 Antibody (TFE3/8663R) - Azide and BSA Free

100 μg

NBP3-21006

Reactivity:

Hu

Species Glossary

Reactivity: Hu

Applications:

Applications:

Host:

Rabbit Monoclonal

Host: Rabbit Monoclonal

Conjugate:

Unconjugated

Available Conjugates:

Conjugate	Catalog #	Availability	Size	Price
Alexa Fluor 350	NBP3-21006AF350
Alexa Fluor 405	NBP3-21006AF405
Alexa Fluor 488	NBP3-21006AF488
Alexa Fluor 532	NBP3-21006AF532
Alexa Fluor 594	NBP3-21006AF594
Alexa Fluor 647	NBP3-21006AF647
Alexa Fluor 700	NBP3-21006AF700
Alexa Fluor 750	NBP3-21006AF750
Allophycocyanin	NBP3-21006APC
Biotin	NBP3-21006B
CoraFluor 1	NBP3-21006CL1
DyLight 350	NBP3-21006UV
DyLight 405	NBP3-21006V
DyLight 488	NBP3-21006G
DyLight 550	NBP3-21006R
DyLight 594	NBP3-21006DL594
DyLight 650	NBP3-21006C
DyLight 680	NBP3-21006FR
DyLight 755	NBP3-21006IR
FITC	NBP3-21006F
HRP	NBP3-21006H
Janelia Fluor 525	NBP3-21006JF525
Janelia Fluor 549	NBP3-21006JF549
Janelia Fluor 585	NBP3-21006JF585
Janelia Fluor 635	NBP3-21006JF635
Janelia Fluor 646	NBP3-21006JF646
Janelia Fluor 669	NBP3-21006JF669
PE	NBP3-21006PE
PerCP	NBP3-21006PCP

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NB100-1028

AIF-1/Iba1 Antibody

(21 Reviews)

(424 Publications)

0.1 mg

NB100-1028

GM1 effects on brain microglia in vivo, after peripheral administration of LPS. Immunoblot in whole brain lysates and densitometric analysis show LPS-dependent increase in AIF-1/Iba1 expression. Image collected and cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/34991625/) licensed under a CC-BY license.

Reactive microglia (green) in peritumoral area of IC injected human glioblastoma cells in mouse brain. NeuN (red), DAPI (blue). Antibody at 1:250. IHC image submitted by a verified customer review.

AIF-1/Iba1 in canine brain. Primary antibody used at 1 ug/mL and incubated with tissue for 30 minutes at room temperature. IHC-P image submitted by a verified customer review.

Staining in mouse brain using AIF-1/Iba1 Antibody. IHC-P image submitted by a verified customer review.

Brown arrow= AIF-1/Iba1 positive cells, Green arrow= mTOR positive cells, Black arrow= mTOR and AIF-1/Iba1 positive cells. IHC-P image submitted by a verified customer review.

Staining in rat hippocampus using AIF-1/Iba1 Antibody. IHC-Fr image submitted by a verified customer review.

Feline optic nerve microglia were immunolabeled with AIF-1/Iba1 Antibody (green), DAPI (blue). Imaging was performed using an epifluorescence microscope. IHC-Fr image submitted by a verified customer review.

Mouse dorsal root ganglia. AIF-1/Iba1 expression in reactive macrophages. Maximum intensity z-projection from 3D images. IHC image submitted by a verified customer review.

Staining in mouse brain using AIF-1/Iba1 Antibody. IHC image submitted by a verified customer review.

Analysis of LPS injected mouse brain tissue, 20 um thickness, frozen. Primary antibody dilution, 1:200. Secondary antibody CY3. Mounted with DAPI mount. Image from verified customer review.

AIF-1/Iba1 staining of AD mouse model (hippocampus). Image from verified customer review.

Negative Control showing staining of paraffin embedded Human Spleen, with no primary antibody.

(0.1ug/ml) staining of Mouse Lymph node lysate (35ug protein in RIPA buffer). Detected by chemiluminescence.

(1ug/ml) staining of Human Frontal Cortex (A) and (0.1ug/ml) Mouse (B) and Rat (C) Brain lysate (35ug protein in RIPA buffer). Detected by chemiluminescence.

(8ug/ml) staining of paraffin embedded Human Spleen. Heat induced antigen retrieval with citrate buffer Ph 6, HRP-staining.

Immunohistochemistry-Paraffin: Goat Polyclonal AIF-1/Iba1 Antibody [NB100-1028] - AIF-1/Iba1 immunoreactivity in an FFPE section of mouse spleen. NB100-1028 was diluted to 1ug per mL and was left on tissue sections for 30m at room temperature. Secondary was horse anti goat HRP polymer. Image from a verified customer review.

Immunohistochemistry-Paraffin: Goat Polyclonal AIF-1/Iba1 Antibody [NB100-1028] - AIF-1/Iba1 immunoreactivity in an FFPE section of human tonsil. NB100-1028 was diluted to 1ug per mL and was left on tissue sections for 30m at room temperature. Secondary was horse anti goat HRP polymer. Image from a verified customer review.

Immunohistochemistry-Paraffin: Goat Polyclonal AIF-1/Iba1 Antibody [NB100-1028] - AIF-1/Iba1 immunoreactivity in an FFPE section of equine spleen. NB100-1028 was diluted to 1ug per mL and was left on tissue sections for 30m at room temperature. Secondary was equine anti goat HRP polymer. Image from a verified customer review.

Immunocytochemistry/ Immunofluorescence: AIF-1/Iba1 Antibody [NB100-1028] - Microglia proliferate in non‐APP mice upon peripheral immune challengeA, BIba‐1, Ki67, & DAPI staining in the cortex & hippocampus of wild‐type & Nlrp3−/− (5 & 15 months old). Microglia proliferate upon LPS injection (white arrows). Non‐microglia cells proliferation was observed as well (yellow arrows). Scale bar: 20 μm.CQuantification of microglial proliferation in cortex (left panel) & hippocampus (right panel) (mean of 5 ± SEM; two‐way ANOVA followed by Tukey's post hoc test, *P 1.5, FDR-corrected p1.5, FDR-corrected p<0.05) to identify GO terms, including inflammatory response (b). The volcano plot shows significant DEGs derived from the NanoString analysis of the brains of Plcg2 inactivation (PLCG2 inact, n=3) & WT (n=3) mice (1-month male) (c). Immunostaining of microglial markers with IBA1 (green) & P2RY12 (red) & quantification of percent area in brain sections of 1-month-old male WT & PLCG2 inact demonstrate unchanged microglial morphology & area coverage (d). GO gene ontology, BP biological process, DEGs differentially expressed genes, FDR false discovery rate Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/35180881), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Immunocytochemistry/ Immunofluorescence: AIF-1/Iba1 Antibody [NB100-1028] - Representative & quantitative immunohistochemistry of the peri-infarct cortex before the bregma (coordinate + 0.58) in normal WT, normal sEH KO, MCAO WT, MCAO sEH KO, MCAO with vehicle treatment & MCAO with AUDA treatment mice (n = 2 for normal groups, n = 5–6 for all groups subjected to MCAO). (A–F, S) The number of CD31-positive endothelial cells significantly increased in the sEH KO before & after MCAO & post-MCAO AUDA-treated mice, while (G–L) Note the increased phosphorylated TrkB (p-TrkB) immunoreactivity that partially co-localized with NeuN-positive neurons, particularly in the sEH KO & AUDA-treated mice (arrows) (T). The percentage of NeuN- & p-TrkB-positive double labeling in the neuronal population was significantly increased compared to that in the corresponding controls. (M–R, U–V) GFAP-positive astrogliosis & Iba1-positive microglial infiltration decreased in post-MCAO sEH KO & AUDA treatment groups relative to that in the corresponding controls. The scale bar represents 50 µm. *p < 0.05, **p < 0.01, ***p < 0.001. Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/29311641), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Immunocytochemistry/ Immunofluorescence: AIF-1/Iba1 Antibody [NB100-1028] - Norgestrel reduces pro-inflammatory & promotes anti-inflammatory phenotypes in rd10 microglia.(A) RT-qPCR analysis detected similar levels of mRNAs encoding progesterone receptor membrane complexes 1 & 2 (PGRMC1, PGRMC2) & all three membrane progesterone receptor isoforms, alpha , beta & gamma (mPR alpha , mPR beta , mPR gamma ) in rd10 retinal microglial cells. Classical progesterone receptor (PR A/B) was not expressed. Actin, HPRT & GAPDH served as reference genes. (B) Immunolabeling confirmed the presence of these progesterone receptors in primary rd10 microglial cells. Hoechst (blue) staining reveals the cell nuclei. Scale bar represents 10μm. (C) Fluorescent microscopic images of primary rd10 microglia immunolabeled for (C) (i) markers of activation (CD68) & pro-inflammation (M1) (iNOS) & (C) (ii) anti-inflammatory activation (M2) (MRC1); after treatment with 20 μM Norgestrel over 24h. Norgestrel reduced pro-inflammatory & increased anti-inflammatory markers in rd10 microglia. Scale bar 10μm. (D) Quantification of fluorescence intensity of (D) (i) CD68 & iNOS & (D) (ii) MRC1 in primary rd10 microglial cultures following Norgestrel treatment over 24h. N = 8 mice, n = 4 technical replicates per group. (E) Nitrite concentration in the media of rd10 microglia in vitro treated with Norgestrel or vehicle over 24hr. Norgestrel significantly reduced nitrite release from rd10 microglia. N = 8 biological replicates, n = 2 technical replicates per sample. Results are presented as mean ± SEM (t-test, *p<0.05, ***p<0.005, ****p<0.001). Image collected & cropped by CiteAb from the following publication (//dx.plos.org/10.1371/journal.pone.0165197), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Immunohistochemistry: AIF-1/Iba1 Antibody [NB100-1028] - Temporal activation of caspase-8 in Iba1-positive myeloid cells. Frozen tissue sections from mice upon pMCAO (n = 9) were analyzed by immunohistochemistry for expression of active caspase-8 or for expression of the microglia/macrophage marker Iba1. The contralateral side to the ischemic area was used an internal control for each animal. A small increase in body size of Iba1 positive cells was detected in the peri-infarct area at 6 h, with larger increase in cell body & numbers of cells expressing Iba1 detected at 24 & 48 h post occlusion (a). Cleaved caspase-8 was detected at low levels at 6 h, but a significant increase in expression levels was observed at 24 & 48 h in the peri-infarct area, but not at the contralateral side (a). Double immunofluorescent staining revealed that increased active caspase-8 expression was localized in Iba1-positive cells (b). Scale bar represent 100 μm in IHC images (a) & 10 μm in IF images (b) Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/27566702), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: AIF-1/Iba1 Antibody [NB100-1028] - GM1 effects on brain microglia in vivo, after peripheral administration of LPS. A Schematic representation of in vivo administration of GM1 after LPS-induced systemic inflammation in mice (N = 5 per treatment). B Representative images of Iba-1 stained microglia in the cortex of control animals infused with vehicle (CTRL) or GM1 (CTRL + GM1), & in animals treated with LPS & subsequently infused with vehicle artificial cerebrospinal fluid (LPS) or GM1 (LPS + GM1). C Immunoblot in whole brain lysates & densitometric analysis show LPS-dependent increase in Iba 1 expression. D Cortical & striatal microglia (Iba-1+ cells) cell body area quantified with MetaXpress software. GM1 administration significantly decreases cell body area in the cortex, but not in the striatum. E IL-6 protein levels in whole mouse brain homogenate. aCSF, artificial cerebrospinal fluid. Bars show mean values ± STDEV. Two-way ANOVA with Tukey’s multiple comparisons test; *p < 0.05, **p < 0.01, ***p < 0.001 Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/34991625), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Immunocytochemistry/ Immunofluorescence: AIF-1/Iba1 Antibody [NB100-1028] - ABIN1 knockdown increases proinflammatory cytokine production & microglial activation. (a-b) Levels of the ABIN1 mRNA & protein were detected 24 h after reperfusion using RT-qPCR & western blot, respectively, to confirm the efficiency of ABIN1 gene knockdown (n = 5 rats per group). (c) ELISA was used to detect the concentrations of TNF-alpha , IL-1 beta & MCP-1 in the peri-infarct cortex at 24 h after reperfusion (n = 5 rats per group). (d) Microglial morphology was observed using immunofluorescence staining for Iba-1 (green) in the peri-infarct cortex at 24 h after reperfusion (n = 3 rats per group). Scale bar = 50 μm. (e & f) Quantification of microglia process endpoints & cell & process lengths/cell (∗∗P < 0.01 compared to the sham group; ##P < 0.01 compared to the MCAO/R group). Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/33110436), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Immunocytochemistry/ Immunofluorescence: AIF-1/Iba1 Antibody [NB100-1028] - SCF+G-CSF treatment increases TREM2 expression in the Iba1+ microglia/macrophages surrounding the 6E10+ senile plaques. (A) Representative confocal images of TREM2 (red), 6E10 (purple) & Iba1 (green) triple immunofluorescence staining in the brains of aged APP/PS1 mice. Blue: Nuclear counterstaining by DAPI. (B) Representative orthographic view of z-stack images (12 z-stacks with 1μm intervals) illustrates the location & interaction of TREM2 + cells (red) & 6E10+ A beta plaques (white) in the brains of aged APP/PS1 mice. (C) Quantification data show the percentage of TREM2+ area surrounding the 6E10+ A beta plaques (within 10μm from the border of the A beta plaques) in the brains of aged APP/PS1 mice with or without SCF+G-CSF treatment. (D) Representative orthographic view of z-stack images (12 z-stacks with 1μm intervals) displays the location & interaction of TREM2+/Iba1+ co-expressing cells (yellow) & 6E10+ A beta plaques (white) in the brains of APP/PS1 mice. (E) Quantification data show the percentage of TREM2+/Iba1+ co-expression area in the total of Iba1+ area in the vicinity of 6E10+ A beta plaques in the brains of aged APP/PS1 mice with or without SCF+G-CSF treatment. N=4-5. Mean ± SEM. * p<0.05 by Student’s t-test. Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/33269098), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Immunocytochemistry/ Immunofluorescence: AIF-1/Iba1 Antibody [NB100-1028] - SCF+G-CSF treatment increases the association of Iba1+ microglia/macrophages with senile plaques & enhances uptake of 4G8+ A beta by the Iba1+ microglia/macrophages in the brains of aged APP/PS1 mice. (A) Representative confocal images illustrate the association of Iba1+ cells (green) with 4G8+ A beta plaques (red) in the brains of aged APP/PS1 mice. Blue: Nuclear counterstaining by DAPI. (B) Quantification of the percentage of Iba1+ area in/surrounding the 4G8+ A beta plaques in the brains of aged APP/PS1 mice with or without SCF+G-CSF treatment. (C) Representative 3-dimensional projected images reveal the overlapped (yellow) Iba1+ cells (green) with 4G8+ A beta (red) in the brains of aged APP/PS1 mice. The white arrows indicate the co-expression (yellow) of 4G8+ A beta & Iba1+ cells in the brains of aged APP/PS1 mice. (D & E) Representative orthographic view of z-stack images (12 z-stacks with 1μm intervals) shows the co-expression (yellow) of 4G8+ A beta (red) & Iba1+ cells (green) in the brains of aged APP/PS1 mice. (F) Quantification data present the percentage of 4G8+ A beta volume within the Iba1+ cells in the brains of aged APP/PS1 mice treated with or without SCF+G-CSF. N=4-5. Mean ± SEM. *p<0.05 by Student’s t-test. Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/33269098), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Immunocytochemistry/ Immunofluorescence: AIF-1/Iba1 Antibody [NB100-1028] - Antibody-mediated Nogo-A blockade decreases the number of cells expressing TNF alpha without affecting the number of Iba1-positive monocytes.Cells expressing TNF alpha , Iba1, & CD68 were labeled on cryosections by immunofluorescence in 4 mouse retinae, 24 h after control IgG or 11C7 injection. a Retinal sections showed many TNF alpha -containing cells located in the inner retina, between the outer plexiform layer & the ganglion cell layer 1 day after control IgG injection. A majority of cells was positive for Iba1 & exhibited vesicular staining of the lysosomal protein CD68, indicating their active state. b The injection of 11C7 strongly attenuated TNF alpha & CD68 expression & to a lower extent the intensity of Iba1 in macrophages/microglia. c Quantification of the number of cells expressing TNF alpha & Iba1 suggesting potent & specific effects of 11C7 on TNF alpha cytokine reduction. Six sections were examined for each retina. Statistics: one-way ANOVA, Dunnett’s post hoc test, **P < 0.01; ****P < 0.0001. Scale bars = 50 μm. Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/32029703), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Immunocytochemistry/ Immunofluorescence: AIF-1/Iba1 Antibody [NB100-1028] - The presence or absence of TRPV1 influences microglial pathophysiological changes in recurrent febrile seizures mice. (A) Images of the cortex immunostained for the microglial marker Iba-1 (visualized in green FITC) & the lysosomal marker CD68/ED1 (visualized in red Alexa Fluor 594) & DAPI (visualized in blue) 5 days after rFS from wild type (WT) & TRPV1−/− mice (Scale bars: 50 μm). (B,C) Bar graph of quantification of Iba1+(B) & Iba1+ ED1+(C) in 5 days after rFS mice cortex. (D) Representative photomicrographs of different microglial morphological subtypes including resting/ramified (Ram), hypertrophic/intermediate (Inter) & round/ameboid (Amoeboid; Scale bars: 5 μm). (E) Quantification of relative percentage of microglia with different morphologies in the cortex of WT & TRPV1−/− mice (n = 6 per groups, **p < 0.01, ***p < 0.001, two-way ANOVA followed by Tukey’s multiple comparisons). (F–H) Representative Immunoblot bands (F) & densitometric analysis of Iba1 expression (G) & ED1 expression (H) were significantly increased in rFS from WT mice than in TRPV1−/− mice. Data were presented as means ± SEM, n = 6 per groups, *p < 0.05, **p < 0.01, ***p < 0.001, two-way ANOVA followed by Tukey’s multiple comparisons. Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/31680864), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

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NB100-449

HIF-1 alpha Antibody

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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

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

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, , , , , , , , ,

Applications: , , , , , , , , ,

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Host: Rabbit Polyclonal

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NBP1-40256

ATF6 Antibody (70B1413.1) - BSA Free

(3 Reviews)

(246 Publications)

0.1 mg

NBP1-40256

$<b>Biological Strategies Validation. </b>Western Blot: ATF6 Antibody (70B1413.1) [NBP1-40256] - Analysis of ATF6 in mouse liver tissue using 3 ug/ml of ATF6 antibody and 0.25 ug/ml of GAPDH antibody. Lane A contains 20 ugs of whole mouse liver lysate, lane B contains 20 ugs of total ER fraction, and lane C contains 20 ugs of rough ER fraction. The ATF6 band may represent under glycosylated or cleaved/active ATF6.$

Immunocytochemistry/Immunofluorescence: ATF6 Antibody (70B1413.1) [NBP1-40256] - Untreated HeLa cells were fixed in -20C methanol for 10 min, air dried and rehydrated in PBS at room temperature for 5 minutes. Cells were incubated with anti-ATF6 (1:20) for one hour at room temperature. ATF6 reactivity (green) was detected with anti-mouse Dylight-488 secondary antibody. Nuclei were counterstained with DAPI (blue). Note the ER localization of ATF6.

Immunohistochemistry-Paraffin: ATF6 Antibody (70B1413.1) [NBP1-40256] - Analysis of ATF6 antibody on Human placenta. Fixed paraffin-embedded sections. Antibody dilution 1:50. Incubated overnight in 4C. Image from verified customer review.

Flow (Intracellular): ATF6 Antibody (70B1413.1) [NBP1-40256] - An intracellular stain was performed on HeLa cells with ATF6 Antibody (70B1413.1) NBP1-40256F (blue) and a matched isotype control (orange). Cells were fixed with 4% PFA and then permeablized with 0.1% saponin. Cells were incubated in an antibody dilution of 10 ug/mL for 30 minutes at room temperature. Both antibodies were conjugated to FITC.. Using the FITC format of this antibody.

Western Blot: ATF6 Antibody (70B1413.1) [NBP1-40256] - Lane 1: 293 cells transfected with full-length ATF6. Lane 2: 293 cells transfected with partial length ATF6 (amino acids 1-373). Lane 3: Untransfected 293 cells. Western blots were probed with 4 ug/ml of the ATF6 monoclonal antibody and visualized with PicoTect Western Blot Chemiluminescence Substrate (10087K). Film was exposed for 1 min. The top arrow corresponds to the 90 kDa form of ATF6 described as full-length in the literature. The human full-length and partial length ATF6 plasmids are described in Luo and Lee (2002).

Immunohistochemistry-Paraffin: ATF6 Antibody (70B1413.1) [NBP1-40256] - Human placenta, followed by biotinylated horse anti-mouse IgG secondary antibody, alkaline phosphatase-streptavidin and chromogen. Dilution 10ug/ml

Flow Cytometry: ATF6 Antibody (70B1413.1) [NBP1-40256] - Intracellular flow cytometric staining of 1 x 10^6 MCF-7 cells using ATF6 antibody (dark blue). Isotype control shown in orange. An antibody concentration of 1 ug/1x10^6 cells was used.

Western Blot: ATF6 Antibody (70B1413.1) [NBP1-40256] - Lysate of DU145 cells. Image from verified customer review.

Western Blot: ATF6 Antibody (70B1413.1) [NBP1-40256] - Lysate of PC-3 cells. Image from verified customer review.

Expression of ER stress-associated transcription factors in NASH, NAFL and normal liver tissues beta -Actin was used as an internal control. Horizontal lines represent means of densitometry signals from the western blot analyses for all tissue groups. #, significant differences in signals between NASH and normal liver tissues (P < 0.05). Data for NAFL tissues were not used for statistical comparisons because of limited sample number (n = 2).

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: ATF6 Antibody (70B1413.1) - BSA Free [NBP1-40256] - The ISR is active in the retinas of rd16 mice.Western blots & corresponding graphs of ISR markers in the retinas of rd16 mice at P15 (a) & P20 (b). At P15, p-eIF2 alpha , ATF4, CHOP, & TRB3 are significantly elevated. At P20, levels of p-eIF2 alpha , ATF4, CHOP, & GADD34 are increased. Markers of the UPR are elevated in rd16 retinas. BiP & cleaved (~50 kD) ATF6 are upregulated in the retinas of rd16 mice at P15 (c) & P20 (d). Relative density measurements correspond to the intensities of the immunoblotting bands or lanes normalized to an internal control. Data are shown as mean ± SEM. a.u. arbitrary units. P15 n = 3. P20 n = 4. *p < 0.05, **p < 0.01, ***p < 0.001 Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/29706649), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: ATF6 Antibody (70B1413.1) - BSA Free [NBP1-40256] - Expression of ER stress-associated transcription factors in NASH, NAFL & normal liver tissues beta -Actin was used as an internal control. Horizontal lines represent means of densitometry signals from the western blot analyses for all tissue groups. #, significant differences in signals between NASH & normal liver tissues (P < 0.05). Data for NAFL tissues were not used for statistical comparisons because of limited sample number (n = 2). Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/28968997), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: ATF6 Antibody (70B1413.1) - BSA Free [NBP1-40256] - Restoration of Plk3 induction rescues ATF2 phosphorylation & ER stress resolution in Lrh-1LKO mice, & loss or gain of ATF2 transcriptional activity also alters ER stress resolution capacity.(A) Primary hepatocytes prepared from Lrh-1f/fand Lrh-1LKO mice & transduced with Ad-Plk3 or Ad-control at a MOI of 100. Cells treated 36 hr later with vehicle or tunicamycin (TM) (0.01 µg/ml) & doxycycline (1 µg/ml) to induce Plk3 or LacZ control. Nuclear protein was obtained at timepoints indicated & immunoblotted for UPR transcription factors, with TBP as a loading control. Results representative of 3 independent experiments. Image collected & cropped by CiteAb from the following publication (//elifesciences.org/articles/01694), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: ATF6 Antibody (70B1413.1) - BSA Free [NBP1-40256] - APPswe induction of OB-senescence via ER stress.a Heat map of differentially expressed ER stress or anti-stress related genes identified by RNA-seq in control (OCN-Cre; Ai9) & TgAPPsweOCN; Ai9 Td+ OB-progenitors (detail analysis was described in Methods). b RT-PCR analysis of ER stress-related genes Grp78, Atf6, Hsp90b1, Eif2ak3, Ern1, Hsp90aa1, & Hspa2 & anti-stress related gene Sirt3 gene expression in purified Td+ BMSCs from 6-MO control (OCN-Cre; Ai9) & TgAPPsweOCN; Ai9 mice, *p < 0.05, **p < 0.01, ***p < 0.001, mean ± SD, n = 3, Mann–Whitney U test. c Western blot analysis of indicated protein expression in BMSCs from mice with indicated genotypes (at 6-MO). GAPDH was used as a loading control. d Quantification of data in c, *p < 0.05, **p < 0.01. mean ± SD, n = 4, Student’s t test. e Western blot analysis of indicated protein expression in BMSCs from 6-MO control & TgAPPsweOCN with or without 0.25 mM 4-PBA (4-Phenylbutyric acid) treatment. f Quantification analyses of the data in e, *p < 0.05, n = 3. g SA-beta -gal staining of 6-MO control & TgAPPsweOCN BMSCs with vehicle (Veh)(PBS) & 4-PBA treatment, respectively, scale bar, 20 µm. h Quantification of SA-beta -gal+ cell densities in g (mean ± SD; n = 5, **p < 0.01, ***p < 0.001). Two-way analysis of variance test was used in f & h. Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/34824365), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: ATF6 Antibody (70B1413.1) - BSA Free [NBP1-40256] - Disturbance of ER homeostasis in ADTKD-UMOD.(A) ADTKD-UMOD is characterized by maturation & trafficking defect of mutant UMOD & intracellular accumulation of UMOD in TAL cells. UMOD immunolocalization revealed a diffuse cytoplasmic staining with enforcement of the luminal membrane in TAL cells of a wild-type mouse. In contrast, TAL cells of an UmodC93F mutant mouse displayed a strong paranuclear immunopositivity for UMOD. Wild-type: Umodwt mouse; UmodC93F: homozygous UmodC93F mutant mouse. Age of mice analysed: four months. Chromogen: DAB, nuclear staining: haemalum. (B) Heat map of relative expression values (z scores) showed differential abundance of several proteins localized in the ER. (C) In the outer medulla of Umod mutant mice of both mouse lines, a strong accumulation of immature UMOD was present. (D) Protein abundances of BiP, phospho-eIF2 alpha , eIF2 alpha , ATF4, both full-length (§) & cleaved activated (#) ATF6, & CHOP were increased in Umod mutant mice compared to wild-type mice. Signal intensities were corrected for GAPDH signal intensities of the same PVDF-membrane, which was stripped several times to facilitate the detection of multiple proteins. Mean of protein abundance of wild-type mice was set on a value of 1 [mean (wild-type) = 1]. Data are shown as means ± SD. One-way ANOVA with Newman-Keuls’s post hoc test: p vs. wild-type, *p < 0.05; **p < 0.01; ***p < 0.001. Image collected & cropped by CiteAb from the following publication (//www.nature.com/articles/srep42970), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: ATF6 Antibody (70B1413.1) - BSA Free [NBP1-40256] - The effect of Ezetimibe on unfolded protein response (UPR) gene expression in THP-1 cells exposed to ischemia-reperfusion (IR). (a) The mRNA expression of activating transcription factor 6 (ATF6) & CCAAT-enhancer-binding protein homologous protein (CHOP). (b) Representative Western blot analyses for the indicated proteins. (c,d) The average quantification of ATF6 & CHOP obtained by the densitometric analysis of three independent experiments. mRNA was analyzed by quantitative real-time PCR; normalized gene expression levels are given as the ratio between the mean value for the target gene & that for beta -actin in each sample. Data are expressed as mean ± SD. * p < 0.01 vs. control (up-regulation); ** p < 0.01 vs. IR; §p < 0.01 vs. control (down-regulation). Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/32340270), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Immunocytochemistry/ Immunofluorescence: ATF6 Antibody (70B1413.1) - BSA Free [NBP1-40256] - Effect of rapamycin on markers of the UPR pathways(A) Cells were incubated in medium alone (0.05% FBS), with 3 nM IGF-1 or with both 3 nM IGF-1 & 10 nM rapamycin (IGF-1 + rapamycin). Total protein extracts prepared from cells incubated for 24 h in those conditions were subjected to Western Blot analysis. Protein expression levels were assessed for phosphorylated or total forms of PERK, eIF2 alpha & for CHOP, BiP, XBP1-s & XBP1-u proteins. Efficiency of rapamycin to inhibit mTORC1 pathway was also checked by immunoblot with phosphorylated & total forms of p70S6K1 & 4E-BP1. alpha -tubulin was used as internal control. (B & C) Cells were incubated with 10nM rapamycin for 1 h (B) or 24 h (C). Immunoblots for phosphorylated & total forms of PERK, eIF2 alpha , p70S6K1 & 4E-BP1 or for ATF4, CHOP, BiP, XBP1-s & XBP1-u protein expression were performed. alpha -tubulin was used as internal control. Blots of P-p70S6K1, p70S6K1, P-PERK, PERK, & alpha -tubulin of Figure 4B & blots of Figure 4C have been performed on the same electrophoresis gel, but cut & reconstituted. (D) Cells were incubated in medium alone (Ctrl) or with 10 nM rapamycin for 24 h. Nuclear localization of ATF6 was assessed using immunofluorescence with ATF6-antibody (red) & Hoechst dye. Magnification ×1000. (E) Bar graphs were obtained by quantification of ATF6 nuclear staining. Results are representative of at least 3 experiments. Image collected & cropped by CiteAb from the following publication (//www.oncotarget.com/lookup/doi/10.18632/oncotarget.15469), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Immunocytochemistry/ Immunofluorescence: ATF6 Antibody (70B1413.1) - BSA Free [NBP1-40256] - Activation of the UPR during hypoxia or glucose depletionSTC-1 & GluTag cells were subjected to hypoxia (1%) or cultivated with decreasing concentration of glucose i.e. 25, 5 or 1 mM, for 24 h. (A) & (C) Protein expression level of phosphorylated & total forms of PERK, eIF2 alpha & ATF4, CHOP, BiP & C-Caspase 3 protein expression was examined using immunoblots, during hypoxia (A) & glucose depletion (C). alpha -tubulin was used as internal control. Blots of Figure 2A have been performed on the same electrophoresis gel, but cut & reconstituted. (B) & (D) XBP1 mRNA splicing was analyzed by RT-PCR after Pst1 digestion: XBP1-u, unspliced; XBP1-h, hybrid; XBP1-s, spliced variant of XBP1; *, XBP1-u mRNA fragments after Pst1 digestion. (E) ATF6 nuclear localization was assessed in STC-1 cells using immunofluorescence with anti-ATF6 antibody & hoechst dye. Magnification x1000. Results are presentative of at least 3 independent experiments (A–E). (F) Bar graphs were obtained by quantification of ATF6 nuclear staining (*P < 0.05 versus control). Results are presentative of 3 independent experiments (A–E) or the mean ± S.E.M. of an experimental n = 3 (F). Image collected & cropped by CiteAb from the following publication (//www.oncotarget.com/lookup/doi/10.18632/oncotarget.15469), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Immunocytochemistry/ Immunofluorescence: ATF6 Antibody (70B1413.1) - BSA Free [NBP1-40256] - UPR status in STC-1 & GluTag cell lines & effect of UPR inducers on markers of the UPR pathwaysSTC-1 & GluTag cells were incubated in medium (Ctrl) or ER stress-inducing agents thapsigargin (Tg, 300 nM), tunicamycin (Tn, 0.05 μg/mL) & brefeldin A (Bref A, 3 μM) for 4 h, 16 h & 8 h respectively. (A) Protein expression level of phosphorylated or total forms of PERK, eIF2 alpha & CHOP, BiP & cleaved-caspase 3 (C-Caspase 3) protein expression was examined using Western Blot analysis. alpha -tubulin was used as internal control (B) Densitometric quantification of P-PERK/PERK, P-eIF2 alpha /eIF2 alpha & BiP/ alpha -tubulin ratios analysis in STC-1 or GluTag cell lines (*P < 0.05 versus control). (C–D) The effect of Tg, Tn & Bref A on ATF6 nuclear localization was assessed by immunofluorescence in STC-1 cells (C) & GluTag cells (D) using anti-ATF6 antibody & Hoechst dye. Magnification ×1000. (E) Bar graphs obtained by quantification of ATF6 nuclear staining (*P < 0.05 versus control). (F) XBP1 mRNA splicing was analyzed by RT-PCR after Pst1 digestion: XBP1-h, hybrid, XBP1-u, unspliced; XBP1-s, spliced variant of XBP1; *, XBP1-u mRNA fragments after Pst1 digestion. Results are presentative of 3 independent experiments (A, C, D, F) or the mean ± S.E.M. of an experimental n = 3 (B, E). Image collected & cropped by CiteAb from the following publication (//www.oncotarget.com/lookup/doi/10.18632/oncotarget.15469), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: ATF6 Antibody (70B1413.1) - BSA Free [NBP1-40256] - Tm‐induced UPR increases expression of RANKL in cultured osteoblastic & osteocytic cells. (A) Western blotting of cell lysates obtained from neonatal calvaria‐derived osteoblastic cells (Calvaria Ob) treated with 2.2 µg/mL Tm for the indicated times. (B‐D) Gene expression as determined by qRT‐PCR in (B) calvaria‐derived osteoblastic cells (n = 3/group) (C) Osteoblastic UAMS‐32 cells (UAMS‐32 Ob) (n = 4/group) or (D) osteocytic MLO‐Y4 cells (MLO‐Y4 Ot) (n = 4/group),maintained in presence of vehicle (, 0.1% DMSO) or 2.2 µg/ml Tm () for 4 hours. (E) Western blot of RANKL protein in cell lysates obtained from calvaria‐derived osteoblastic cells as described in A in a separate study. Data shown are the mean & SD with individual data points. *P < .05 vs vehicle by Student's t‐test Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/32259048), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Reactivity:

Hu, Mu, Rt, Po, Pl, Rb

Species Glossary

Reactivity: Hu, Mu, Rt, Po, Pl, Rb

Applications:

, , , , , , , ,

Applications: , , , , , , , ,

Host:

Mouse Monoclonal

Host: Mouse Monoclonal

Alt Catalog#

IMG-273

Alt Catalog# IMG-273

Sizes:

Sample Size Available

Sizes: Sample Size Available

Conjugate:

Unconjugated

Available Conjugates:

Validated by:

Validated by:

Conjugate	Catalog #	Availability	Size	Price
Alexa Fluor 350	NBP1-40256AF350
Alexa Fluor 405	NBP1-40256AF405
Alexa Fluor 488	NBP1-40256AF488
Alexa Fluor 532	NBP1-40256AF532
Alexa Fluor 594	NBP1-40256AF594
Alexa Fluor 647	NBP1-40256AF647
Alexa Fluor 700	NBP1-40256AF700
Alexa Fluor 750	NBP1-40256AF750
Allophycocyanin	NBP1-40256APC
Biotin	NBP1-40256B
CoraFluor 1	NBP1-40256CL1
DyLight 350	NBP1-40256UV
DyLight 405	NBP1-40256V
DyLight 488	NBP1-40256G
DyLight 550	NBP1-40256R
DyLight 594	NBP1-40256DL594
DyLight 650	NBP1-40256C
DyLight 680	NBP1-40256FR
DyLight 755	NBP1-40256IR
FITC	NBP1-40256F-0.1ml
HRP	NBP1-40256H
Janelia Fluor 525	NBP1-40256JF525
Janelia Fluor 549	NBP1-40256JF549
Janelia Fluor 585	NBP1-40256JF585
Janelia Fluor 635	NBP1-40256JF635
Janelia Fluor 646	NBP1-40256JF646
Janelia Fluor 669	NBP1-40256JF669
PE	NBP1-40256PE
PerCP	NBP1-40256PCP
mFluor Violet 450 SE	NBP1-40256MFV450
mFluor Violet 500 SE	NBP1-40256MFV500
mFluor Violet 610 SE	NBP1-40256MFV610

Available Formulations

Formulation	Catalog #	Availability	Price
BSA Free	NBP1-40256SS
Azide and BSA Free	NBP2-80583

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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

HIF-1 alpha Antibody - BSA Free

Transcription factor E3 Overexpression Lysate

Transcription factor E3 Antibody (TFE3/6849R)

Transcription factor E3 Antibody

Transcription factor E3 Antibody

Transcription factor E3 Antibody (TFE3/8663R)

Transcription factor E3 Antibody (TFE3/8663R) - Azide and BSA Free

AIF-1/Iba1 Antibody

HIF-1 alpha Antibody

ATF6 Antibody (70B1413.1) - BSA Free