Western Blot: Novus Biologicals

NB100-2220

LC3B Antibody - BSA Free

(51 Reviews)

(1731 Publications)

0.1 ml

NB100-2220

Lysates of HeLa parental cell line and LC3B knockout HeLa cell line (KO) untreated (-) or treated (+) with 50 uM CQ for 18 hours. PVDF (Polyvinylidene difluoride) membrane was probed with 0.5 ug/mL of Rabbit Anti-LC3B Polyclonal Antibody (Catalog # NB100-2220) followed by HRP-conjugated Anti-Rabbit IgG Secondary Antibody (Catalog# HAF008). A specific band was detected for LC3B at a molecular weight of approximately 15 kDa (as indicated) in the parental HeLa cell line, but is not detectable in the knockout HeLa cell line. GAPDH is shown as a loading control. This experiment was conducted under reducing conditions.

<b>Genetic Strategies Validation. </b>LC3B was detected in immersion fixed CQ treated HeLa cells (left) but was not detected in LC3B knockout HeLa cells (right) using rabbit anti-human LC3B polyclonal antibody (Catalog #NB100-2220) at 0.3 ug/mL for 3 hours at room temperature. Cells were stained using the NorthernLights(TM) 557-conjugated anti-Rabbit IgG Secondary Antibody (red; Catalog # NL004) and counterstained with DAPI (blue). Specific staining was localized to cytoplasm.

<b>Biological Strategies Validation. </b>Contribution of autophagy to lipid droplet formation in WT and KO NSC34 cells.(A) WT and KO NSC 34 cells were treated with (+) or without (-) bafilomycin A (bafA) under basal or HBSS starvation conditions for 4 h and blotted for p62, LC3, and GAPDH. Image collected and cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/32321733) licensed under a CC-BY license.

TMEM16A inhibited AngII-induced autophagy in aortas. (D) Representative immunofluorescence staining of LC3 (green) and a-SMA (red) in aortas. Scale bars, 20 um. Quantification of LC3B puncta. Five random fields (2,500 um2/filed) were measured in one section. **P < 0.01 vs. sham + TMcon; ##P < 0.01 vs. AngII + TMcon, one-way ANOVA. n = 14 sections from seven mice per group. Image collected and cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/32226533) licensed under a CC-BY license.

LC3 accumulation in muscle fibers from patients with PAD (row B - E). Normal fibers from a Non-PAD sample (row A). Scale bar = 100 uM. Image from verified customer review.

Localization of LC3 by immunohistochemical method. The immunohistochemical staining shows that LC3, CRF, and HIF-1alpha have an overlapping localization in villous and extravillous trophoblast. d: decidua; v: villi; DeVe: decidual vessel. Original magnification 40x. Image collected and cropped by CiteAb from the following publication (//www.hindawi.com/journals/bmri/2013/689768/) licensed under a CC-BY license.

Detection of LC3B in mouse ES cell lysates using Rabbit anti- [Catalog # NB100-2220]. LC3 detected at a molecular weight of approximately 17 kDa. Atg5-/- ES cells from Dr. Noboru Mizushima [Mizushima, N. et al. J. Cell Biol. 152 (2001)] Photo courtesy of Dr. Beth Levine, UT SW Medical Center.

ORP1L and VAP-A form cholesterol-dependent ER-AV contact sites MelJuSo cells expressing GFP or GFP-tagged ORP1L mutants were fixed and stained for LC3 and Calnexin. Y477 and D478 in the FFAT motif were mutated to alanines in the ORP1L ydaa mutant. Scale bar, 10 um. Right: co-immunoprecipitation for ORP1L (mutants) with VAP-A. GFP-ORP1L mutants or GFP were isolated from lysates of HEK293T cells co-overexpressing HA-VAPA using GFP-Trap beads. Western blot filters were probed for isolated GFP-tagged proteins, the associated HA-VAP-A and the input HA-VAP-A, as indicated. Image collected and cropped by CiteAb from the following publication (//www.nature.com/doifinder/10.1038/ncomms11808), licensed under a CC-BY license.

<b>Biological Strategies Validation. </b>Lysates of mouse NIH3T3 and rat PC-12 cell lines untreated (-) or treated (+) with CQ. PVDF (Polyvinylidene difluoride) membrane was probed with 0.5 ug/mL rabbit anti-LC3B polyclonal Antibody (Catalog # NB100-2220, Novus Biologicals), followed by 1:2000 dilution of goat anti-rabbit IgG secondary antibody. LC3 detected at a molecular weight of approximately 15 kDa in treated NIH3T3 and PC-12 cells.

<b>Biological Strategies Validation. </b>Detection of LC3I and LC3II in mouse cochlea cell line SV-K1 using Rabbit anti- [Catalog # NB100-2220]. Cells were treated with CQ (1 uM), and As2O3 (1 uM) for 24 hrs. Image from verified customer review.

Autophagy Investigation. (A) Representative WBs (Western Blots) of LC3B-I, LC3B-II and GAPDH loading control in DANs derived from 4 iPD patients and 4 healthy controls. (B) WBs from three independent experiments were quantified by densitometry and LC3B-II normalized to a loading control. Grouping of data by healthy vs iPD as well as iPD stratified for T/T and C/C genotype. (C) Ratio of LC3B-II (normalized to loading control) following bafilomycin treatment/untreated. Citation: Marrone L, Bus C, Schondorf D, Fitzgerald JC, Kubler M, Schmid B, et al. (2018) Generation of iPSCs carrying a common LRRK2 risk allele for in vitro modeling of idiopathic Parkinson's disease. PLoS ONE 13(3): e0192497. //doi.org/10.1371/journal.pone.0192497

<b>Biological Strategies Validation. </b>Expression of autophagic protein LC3 in BME-UV1 cells forming acinar structures on Matrigel. Representative images of Western blot analysis of LC3 in bovine MECs grown in 3D culture for 3, 6, 9, and 14 days in differentiation medium (control), enriched with 17beta-estradiol (E2, 1 nM), progesterone (P4, 5 ng/mL), or both (E + P); expression of beta-actin was used as a loading control; graphs below the images show the results of densitometric analysis, in which IOD of each band was measured, and the values were normalized to IOD of beta-actin; the IOD results are presented as means +/- SEM from at least three separate experiments. Image collected and cropped by CiteAb from the following publication (//www.hindawi.com/journals/bmri/2014/382653/) licensed under a CC-BY license.

GNA triggers the formation of autophagic markers in A549 and HeLa cell. Effects of GNA on LC3 protein. A549 cells were treated with various concentrations of GNA for 24 hours or 3 uM GNA for the indicated periods of time, then analyzed by western blotting using anti-LC3 antibodies. GAPDH protein was used as the loading control. The bar graph shows the band intensities of LC3-II relative to those of GAPDH. Mean+/- SEM, n = 3, *means p<0.05, ** p<0.01, one-way ANOVA. Image collected and cropped by CiteAb from the following publication (//dx.plos.org/10.1371/journal.pone.0083604), licensed under a CC-BY license.

Autophagosomes accumulate in neurons of the VH gray matter at day 1 after SCI. Representative images of IHC staining for LC3 (green) and neuronal marker NeuN (red) in VH of gray matter from sham and SCI animals. Stronger co-localization between NeuN and LC3 is apparent at day 1 after SCI. Scale bar is 20 um. Image collected and cropped by CiteAb from the following publication (//www.nature.com/articles/cddis2014527) licensed under a CC-BY license.

Autophagosomes accumulate in microglia and oligodendrocytes of the dorsal white matter adjacent to injury after SCI. Representative images of IHC staining for LC3 (green) and activated microglia marker CD11B (red) in dorsal white matter of sham and SCI animals. Increased co-localization between LC3 and CD11B is apparent at day 1 after SCI. Image collected and cropped by CiteAb from the following publication (//www.nature.com/articles/cddis2014527) licensed under a CC-BY license.

Staining of rat brain tissue section. Analysis using Rabbit anti-LC3B Antibody [Catalog # NB100-2220]. Image from verified customer review.

Human ovarian Cancer tissue stained using heat mediated antigen retrieval in pH 6.0 citrate buffer at 1:200 dilution. Image provided by verified customer review.

FFPE (Formalin-Fixed Paraffin-Embedded) tissue section of mouse brain using 1:200 dilution of Rabbit anti-LC3B antibody [Catalog # NB100-2220]. The specific signal of LC3 was detected using HRP-conjugated secondary antibody with DAB (3, 3 -diaminobenzidine) reagent, and nuclei of cells were counterstained using hematoxylin. This LC3B antibody generated a low to moderate levels of cytoplasmic staining in the glial cells. The neurons depicted a moderate to strong staining for LC3 in their cytoplasm.

Analysis using the Biotin conjugate of Rabbit anti-LC3B Antibody [Catalog # NB100-2220]. Staining of brain, cerebral cortex, neurons with cell processes.

SCI leads to lysosomal dysfunction. Representative high magnification images ( x 60) of LC3 (green) and CTSD (red) staining in VH of gray matter from sham and day 1 SCI animals. Decrease in number and intensity of CTSD puncta (lysosomes) and increase in LC3 puncta (autophagosomes) is apparent after SCI. Scale bar is 10 um. Image collected and cropped by CiteAb from the following publication (//www.nature.com/articles/cddis2014527) licensed under a CC-BY license.

Image shows a specific band for LC3B at a molecular weight of approximately 15 kDa in 0.5 mg/mL of Neuro2A lysate. This experiment was performed under reducing conditions using the 12-230 kDa separation system.

Reactivity:

Hu, Mu, Rt, Po, Al, Av, Ba, Bv, Ca, Ch, ChHa, Gp, Ha, In, Pm, Pm, Rb, SyHa, Ze

Species Glossary

Reactivity: Hu, Mu, Rt, Po, Al, Av, Ba, Bv, Ca, Ch, ChHa, Gp, Ha, In, Pm, Pm, Rb, SyHa, 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	NB100-2220AF350
Alexa Fluor 405	NB100-2220AF405
Alexa Fluor 488	NB100-2220AF488
Alexa Fluor 532	NB100-2220AF532
Alexa Fluor 594	NB100-2220AF594
Alexa Fluor 647	NB100-2220AF647
Alexa Fluor 700	NB100-2220AF700
Alexa Fluor 750	NB100-2220AF750
Allophycocyanin	NB100-2220APC
Biotin	NB100-2220B
CoraFluor 1	NB100-2220CL1
DyLight 350	NB100-2220UV
DyLight 405	NB100-2220V
DyLight 488	NB100-2220G
DyLight 550	NB100-2220R
DyLight 594	NB100-2220DL594
DyLight 650	NB100-2220C
DyLight 680	NB100-2220FR
DyLight 755	NB100-2220IR
FITC	NB100-2220F
HRP	NB100-2220H
Janelia Fluor 525	NB100-2220JF525
Janelia Fluor 549	NB100-2220JF549
Janelia Fluor 585	NB100-2220JF585
Janelia Fluor 635	NB100-2220JF635
Janelia Fluor 646	NB100-2220JF646
Janelia Fluor 669	NB100-2220JF669
PE	NB100-2220PE
PerCP	NB100-2220PCP
mFluor Violet 450 SE	NB100-2220MFV450
mFluor Violet 500 SE	NB100-2220MFV500
mFluor Violet 610 SE	NB100-2220MFV610

Available Formulations

Formulation	Catalog #	Availability	Price
BSA Free	NB100-2220SS

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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 < 0.05, ***p < 0.001) Image collected and cropped by CiteAb from the following publication (//www.nature.com/articles/s41467-019-10444-1) licensed under a CC-BY license.

Analysis using the biotin conjugate of NB100-105. Staining of human glioblastoma multiforme.

<b>Biological Strategies Validation. </b>HIF-1 alpha was detected in immersion fixed HeLa human cervical epithelial carcinoma cell line treated with DFOA using 1 ug/mL of mouse anti-HIF-1 alpha monoclonal antibody NB100-105. Cells were stained using a donkey anti-rabbit secondary antibody and counterstained with DAPI (blue).

Upregulation of HIF-1alpha in human PCa. Protein expression of HIF-1alpha, VEGF, and GLUT4 were examined with western blot, in PC-3, DU145, and 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-1alpha or DDP group. Original blots are shown in Supplementary Figure 5. C: Ctrl; D: DDP; S: si-HIF-1alpha; D/S: DDP/si-HIF-1alpha. Image collected and cropped by CiteAb from the following publication (//www.nature.com/articles/s41598-017-07973-4), licensed under a CC-BY license.

Analysis using HIF-1 alpha Antibody on human throat cancer tissue. PFA fixed paraffin-embedded sections. Primary antibody dilution 1:100. Image from verified customer review.

<b>Biological Strategies Validation. </b>Analysis of HIF-1 alpha in human hepatocytes from cancer patient using anti-HIF-1 alpha antibody NB100-105. Western blot image submitted by a verified customer review.

<b>Biological Strategies Validation. </b>Analysis using the HRP conjugate of NB100-105. Detection of 50 ug cobalt chloride induced COS-7 nuclear extracts (NB800-PC26) using NB100-105.

L. donovani infection induces HIF-1 alpha expression in CD11chi splenic DCs in an IRF-5 dependent manner. Mice were infected with 2 x 10^7 amastigotes intravenously. Immunoblot analysis of HIF-1 alpha expression in CD11c+ cells from C57BL/6 mice (upper panel) and densitometric analysis normalized to beta-actin expression and expressed as fold increase to results obtained with naive mice (lower panel). Image collected and cropped by CiteAb from the following publication (//dx.plos.org/10.1371/journal.ppat.1004938), licensed under a CC-BY license.

Upregulation of HIF-1alpha in human Pca. HIF-1alpha protein was detected by western blot in nonmalignant (RWPE-1 and BPH1) and PCa cell lines (PC-3, DU145, LNCaP, and 22RV1) as indicated. Image collected and cropped by CiteAb from the following publication (//www.nature.com/articles/s41598-017-07973-4), licensed under a CC-BY license.

HIF-1alpha expression in PDAC and adjacent normal pancreatic tissue. PDAC with weak (intensity 1) nuclear and cytoplasmic HIF-1alpha staining (left). PDAC with strong (intensity 3) nuclear and moderate (intensity 2) cytoplasmic HIF-1alpha staining (right). Image collected and cropped by CiteAb from the following publication (//wjso.biomedcentral.com/articles/10.1186/s12957-018-1432-4), licensed under a CC-BY license.

Analysis using the Alexa Fluor(R) 488 conjugate of NB100-105. Staining of HIF-1 alpha in H929 cells using HIF-1 alpha antibody. Flow cytometry image submitted by a verified customer review.

Effects of one- and five-dosage dry needling (1D, 5D) on iNOS, HIF-1 alpha , COX-2, and VEGF expressions in a needling-treated muscle. (a) Representative Western blot images. The quantification of the protein levels for (b) iNOS, (c) HIF-1 alpha , (d) COX-2, and (e) VEGF. Values are expressed as mean ± SD. *Indicates the significant difference (P < 0.05) between the sham groups (s1D and s5D). #Represents the significant difference (P < 0.05) between the 1D and 5D groups. DN: dry needling.

Hypoxia is implicated in the adaptive response after short-term bevacizumab treatment. Expression of HIF-1 alpha in pulmonary tumor nodules of the four groups. (A) A representative western blot is shown. beta -actin was used as a loading control. (B) While most tumors showed little expression of HIF-1 alpha protein in PBS and cisplatin groups, mice that received bevacizumab and bevacizumab + cisplatin therapy showed a markedly increased level of HIF-1 alpha expression.. *P < 0.05, **P < 0.01.

Effects of F2 on tumor-related biomarkers in vivoImmunohistochemical analysis of tumor-related proteins (HIF-1 alpha , VEGF, MMP-2, CD31 and KI67) in tumor sections from U87 xenograft mice treated with F2 (mg/kg i.g.). Red arrows indicate the related protein detected in the tissue samples. Values are means ± SEM of three experiments. * P<0.05 compared with DDW group.

AAM inhibits hypoxia-induced VM formation involving HIF-1 alpha and MMP2. The HCT-116 (A) and LoVo (B) cells were treated with different concentrations of AAM under hypoxia and normoxia; the protein expression of HIF-1 alpha and MMP2 was detected by western blotting. (C, D) The mRNA expression of HIF-1 alpha and MMP2 was measured by RT-PCR (*P < 0.05, **P < 0.01, ***P < 0.001).

Immunohistochemical analysis of STC1 expression in esophageal cancer. (A) Normal squamous epithelium was negative for STC1 expression. (B) Normal gastric glands showed positive staining for STC1, which was used as a positive control. (C) Positive staining for STC1 in esophageal squamous cell carcinoma (ESCC). (D) Negative staining for STC1 in ESCC. (E) Positive staining for HIF-1 alpha in ESCC. (F) Positive staining for p53 in ESCC. Magnification, ×100.

Simple Western: HIF-1 alpha Antibody (H1alpha67) [NB100-105] - Effect of hypoxia on human airway epithelial cells (A549).A549 cells were subjected to hypoxia for 6, 24, and 48 hours. Cell lysates were collected and analyzed for the expression levels of various hypoxic markers by qPCR (n = 4). (A-C) The expression levels of HIF-1 alpha (A), GLUT-1 (B), and VEGFA (C) were measured. (D-G) Additionally, whole-lung lysate was used for Western blots to determine the protein expression of HIF-1 alpha, VEGFA, and FIH-1. After analyzing the data, we used the unpaired t test with Welch's correction to determine its statistical significance. *P < 0.05, ***P < 0.001, ****P < 0.0001. Image collected and cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/37737265 ), 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.

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

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NB100-304

53BP1 Antibody - BSA Free

(14 Reviews)

(753 Publications)

0.1 mg

NB100-304

Irradiated cochlear spiral ganglion cells, mouse, frozen section of fixed material. Image from verified customer review.

<b>Genetic Strategies Validation. </b>53BP1 was detected in immersion fixed HeLa cells (left) but was not detected in 53BP1 knockout Hela cells (right) using Rabbit Anti-human 53BP1 polyclonal antibody (Catalog #NB100-304) at 0.3 ug/mL for 3 hours at room temperature. Cells were stained using the NorthernLights™ 557-conjugated Anti-Rabbit IgG Secondary Antibody (red; Catalog # NL004) and counterstained with DAPI (blue). Specific staining was localized to nuclei.

Total protein from HeLa, A431, Neuro2A, and PC12 was separated on a 12% gel by SDS-PAGE, transferred to PVDF membrane and blocked in 5% non-fat milk in TBST. The membrane was probed with 1.0 ug/mL in 5% block buffer and detected with an anti-rabbit HRP secondary antibody using chemiluminescence. The observed molecular weight for these samples are ~250 kDa and the theoretical molecular weight is 214 kDa.

Human breast tumors stained with 53BP1 antibody. Image from verified customer review.

Detection of Human and Mouse 53BP1 by IHC. Sample: FFPE sections of human ovarian carcinoma (left) and mouse teratoma (right). Antibody: 53BP1 Antibody (Catalog #NB100-304) used at a dilution of 1:1000 (1ug/mL). Detection: DAB.

Human medulloblastoma (DAOY) and mouse astrocyte (C8D1A) cell lines were exposed for 48 hours to DMSO or 1ug/mL of the DNA damaging agent EPE. Cells were immunostained for 53BP1 (green). The nuclei were counterstained with DAPI (blue). Image from verified customer review.

An intracellular stain was performed on RH-30 cells with 53BP1 Antibody (Catalog #NB100-304AF647) (blue) and a matched isotype control (orange). Cells were fixed with 4% PFA and then permeabilized with 0.1% saponin. Cells were incubated in an antibody dilution of 5 ug/mL for 30 minutes at room temperature. Both antibodies were conjugated to Alexa Fluor 647.

Immunoprecipitation analysis lysates from HCC44 cells in 1% NP40. Image from verified customer review.

<b>Biological Strategies Validation. </b>Effect of RK-33 on radiation-induced DNA damage. A Immunofluorescence images showing 53BP1 and gamma-H2AX foci in A549 cells after 2-Gy radiation and A549 cells pre-treated with 6 uM RK-33, 12 h before radiation. Overlap of 53BP1 and gamma-H2AX is seen in the merged picture of the co-immunofluorescence staining. Scale bar is 2 um. Image collected and cropped by CiteAb from the following publication (//embomolmed.embopress.org/cgi/doi/10.15252/emmm.201404368) licensed under a CC-BY license.

Lamin A/C-53BP1 interaction is regulated in a DNA damage-dependent manner. U2OS/GFP-lamin A cells were pretreated with 10 um ATMi f for 1 h before exposure to IR (10 Gy, 1 h recovery). Cell extracts were then subjected to immunoprecipitation using GFP-Trap beads, and bound complexes were then analyzed by immunoblotting using 53BP1 and GFP antibodies. WCE represents 1% input. Image collected and cropped by CiteAb from the following publication (//doi.wiley.com/10.1111/acel.12258), licensed under a CC-BY license.

HeLa cells were fixed in 4% paraformaldehyde for 10 minutes and permeabilized in 0.5% Triton X-100 in PBS for 5 minutes. The cells were incubated with 53BP1 Antibody conjugated to DyLight 550 (NB100-304R) at 5 ug/ml for 1 hour at room temperature. Nuclei were counterstained with DAPI (Blue). Cells were imaged using a 40X objective.

An intracellular stain was performed on Ntera2 cells with 53BP1 Antibody NB100-304 (blue) and a matched isotype control NBP2-24891 (orange). Cells were fixed with 4% PFA and then permeabilized with 0.1% saponin. Cells were incubated in an antibody dilution of 1.0 ug/mL for 30 minutes at room temperature, followed by Rabbit IgG (H+L) Cross-Adsorbed Secondary Antibody, Dylight 550 (SA5-10033, Thermo Fisher).

Whole cell lysate from U2OS or 293T cells. Bands indicate an observed molecular weight of ~220 kDa and the theoretical molecular weight is 214 kDa.

<b>Biological Strategies Validation. </b>Upper Panel: 53BP1 foci in proliferating MEFs. Lower Panel: 53BP1 foci in proliferating MEFs exposed to 10 Gy of IR.

Embryonic Fibroblast cells pre-extraction for 5 mins with CSK buffer. Fixed with 4% PFA and 75% Ethanol. Primary Antibody at 1:1000. Secondary Antibody at 1:1000. Image from verified customer review.

<b>Biological Strategies Validation. </b>Upper Panel: Control untreated cells. Lower Panel: Cells exposed to irradiation (10 Gy) and probed for 53BP1 foci. Cells were grown on coverslips, fixed with 4% paraformaldehyde, methanol permeabilized, blocked for 1 h, RT. Incubated with primary antibody (1:200) overnight, washed 3x with PBS, probed with tubulin (Alexa Fluor 594) antibody for 2 h, RT. Washed 3x with PBS, mounted on slides using prolong gold, imaged using Nikon confocal microscope (100x oil). Image from verified customer review.

<b>Genetic Strategies Validation. </b>MUS81 inhibition in BRCA2-deficient cells causes accumulation of 53BP1 nuclear bodies and G1 arrest. H1299 cells carrying a DOX-inducible BRCA2 shRNA were transfected with control or MUS81 siRNAs. Representative images of cells processed 72 h later for immunostaining with 53BP1 Antibody (green) and anti-cyclin A (red) antibodies. DNA was counterstained with DAPI. Scale bar, 10 um. Image collected and cropped by CiteAb from the following publication (//www.nature.com/doifinder/10.1038/ncomms15983), licensed under a CC-BY license.

<b>Biological Strategies Validation. </b>Pot1a prevents DDR in HSCs. Telomeric DDR in 8 week-old LSK cells upon Pot1a knockdown. Immunocytochemical staining of TRF1 (green). Foci co-stained with TRF1 and 53BP1 were identified as TIFs. Nuclei were stained with TOTO3 (blue). Scale bar, 2 um. Image collected and cropped by CiteAb from the following publication (//www.nature.com/articles/s41467-017-00935-4), licensed under a CC-BY license.

Lamin A/C-53BP1 interaction is regulated in a DNA damage-dependent manner. U2OS cells were transfected with siCTRL or siLMNA and subjected to laser micro-irradiation, fixed 15 min later and then processed for immunofluorescence with gamma-H2AX and 53BP1 antibodies. Scale bar, 10 um. Image collected and cropped by CiteAb from the following publication (//doi.wiley.com/10.1111/acel.12258), licensed under a CC-BY license.

Ntera2 cells were fixed in 4% paraformaldehyde for 10 minutes and permeabilized in 0.5% Triton X-100 in PBS for 5 minutes. The cells were incubated with anti-53BP1 Antibody NB100-304 at 2 ug/ml overnight at 4C and detected with an anti-rabbit Dylight 488 (Green) at a 1:1000 dilution for 60 minutes. Alpha tubulin (DM1A) NB100-690 was used as a co-stain at a 1:1000 dilution and detected with an anti-mouse Dylight 550 (Red) at a 1:1000 dilution. Nuclei were counterstained with DAPI (Blue). Cells were imaged using a 100X objective and digitally deconvolved.

NIH3T3 cells were fixed in 4% paraformaldehyde for 10 minutes and permeabilized in 0.5% Triton X-100 in PBS for 5 minutes. The cells were incubated with anti-53BP1 Antibody NB100-304 at 2 ug/ml overnight at 4C and detected with an anti-rabbit Dylight 488 (Green) at a 1:1000 dilution for 60 minutes. Alpha tubulin (DM1A) NB100-690 was used as a co-stain at a 1:1000 dilution and detected with an anti-mouse Dylight 550 (Red) at a 1:1000 dilution. Nuclei were counterstained with DAPI (Blue). Cells were imaged using a 100X objective and digitally deconvolved.

Staining of 53BP1 in human colon cancer using DAB with hematoxylin counterstain.

An intracellular stain was performed on A431 cells with 53BP1 Antibody (Catalog #NB100-304AF647) (blue) and a matched isotype control (orange). Cells were fixed with 4% PFA and then permeabilized with 0.1% saponin. Cells were incubated in an antibody dilution of 2.5 ug/mL for 30 minutes at room temperature. Both antibodies were conjugated to Alexa Fluor 647.

For 53BP1 staining, samples were incubated for 1 hour with 1:200 53BP1 primary antibody in PBS/BSA 1%, at room temperature. Green: 53BP Blue: DAPI. Image from verified customer review.

DYNLL1 is required for 53BP1-dependent p53 responses to Nutlin-3. a Immunoblot analysis of the MCF-7 cell lines used in (a) with anti-53BP1 or anti-RPA32 antibodies prior to N3 treatment. b Quantification of n = 3 independent experiments represented in (a), each performed in triplicate. Mean ± SD. c Indicated parental or stably complemented 53BP1-/- MCF-7 cell lines were incubated in the presence (11 days) or absence (7 days) of Nutlin-3 (4 µM)

Depletion of PNUTS and NPAT induces a global DDR.(A) Induction of telomere dysfunction–induced foci (TIF) upon candidate knockdown in HeLa cells from Fig 5. Depletion of TRF2 was used as a positive control. 53BP1 was detected by indirect IF and telomeres by FISH with a Cy3-[CCCTAA]3 probe. (B) Frequency distribution of the number of TIFs per cell from (A). n indicates the number of cells analyzed for each condition and red lines mark the mean.

HPV+ HNSCCs have decreased expression of NHEJ and HR proteins including DNA-Pk and BRCA2Cells were treated with mock or 4 Gy radiation, harvested and lysed at 15 minutes post-treatment, and analyzed by western blot for relative expression of indicated proteins. beta -actin was used as a loading control. Shown is a representative blot from 2 independent experiments.

LNCaP cells in culture were treated for 24 hrs with DMSO or ENZ, then irradiated with 0 or 1 Gy.Cell were fixed and immunostained for 53BP1 0.5, 1.5 and 24 hrs after irradiation. Immunofluorescent images were obtained by confocal microscopy. White bar = 20 microns. A) DMSO alone, B) ENZ alone, C) 1 Gy, D) ENZ + 1 Gy. White bar = 20 microns.

Representative immunohistochemistry staining for 53BP1 expression in pancreatic adenocarcinoma. (A) is high intensity of 53BP1 expression and (B) is low intensity of 53BP1 expression.

53BP1 was detected in immersion fixed U-2 OS human osteosarcoma cell line using Rabbit anti-53BP1 Antigen Affinity Purified Polyclonal Antibody conjugated to DyLight 650 (Catalog # NB100-304C) (light blue) at 10 µg/mL overnight at 4C. Cells were counterstained with DAPI (blue). Cells were imaged using a 100X objective and digitally deconvolved.

Reactivity:

Hu, Mu, Rt, Bt, Bv, Ca, Fi, Gt, Pm, Po, Pm, Fe, Pm-Or, Rb, Sh

Species Glossary

Reactivity: Hu, Mu, Rt, Bt, Bv, Ca, Fi, Gt, Pm, Po, Pm, Fe, Pm-Or, Rb, 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	NB100-304AF350
Alexa Fluor 405	NB100-304AF405
Alexa Fluor 488	NB100-304AF488
Alexa Fluor 532	NB100-304AF532
Alexa Fluor 594	NB100-304AF594
Alexa Fluor 647	NB100-304AF647
Alexa Fluor 700	NB100-304AF700
Alexa Fluor 750	NB100-304AF750
Allophycocyanin	NB100-304APC
Allophycocyanin/Cy7	NB100-304APCCY7
Biotin	NB100-304B
CoraFluor 1	NB100-304CL1
DyLight 350	NB100-304UV
DyLight 405	NB100-304V
DyLight 488	NB100-304G
DyLight 550	NB100-304R
DyLight 594	NB100-304DL594
DyLight 650	NB100-304C
DyLight 680	NB100-304FR
DyLight 755	NB100-304IR
FITC	NB100-304F
HRP	NB100-304H
Janelia Fluor 525	NB100-304JF525
Janelia Fluor 549	NB100-304JF549
Janelia Fluor 585	NB100-304JF585
Janelia Fluor 635	NB100-304JF635
Janelia Fluor 646	NB100-304JF646
Janelia Fluor 669	NB100-304JF669
PE	NB100-304PE
PE/Atto594	NB100-304PEATT594
PE/Cy5.5	NB100-304PECY55
PE/Cy7	NB100-304PECY7
PerCP	NB100-304PCP
mFluor Violet 450 SE	NB100-304MFV450
mFluor Violet 500 SE	NB100-304MFV500
mFluor Violet 610 SE	NB100-304MFV610

Available Formulations

Formulation	Catalog #	Availability	Price
BSA Free	NB100-304SS

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NB600-501

beta-Actin Antibody (AC-15)

(27 Reviews)

(592 Publications)

0.1 ml

NB600-501

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

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

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

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

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

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

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

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

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

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

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

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

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

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

TRIB3 supports MYC stability by disturbing UBE3B-mediated MYC ubiquitination.a Heatmap presenting TRIB3 expression in 11 blood cancer cell lines and 1 primary human lymphoma cell line (T144) with or without TRIB3 depletion. b Effect of TRIB3 overexpression on MYC degradation in vivo. Raji cells were infected with TRIB3- or GFP- adenovirus, and 12 h later, incubated with cycloheximide (CHX; 10 μg/mL) for the indicated times. The data are presented as the means ± S.E.M from three independent experiments. c Effect of TRIB3 deletion on MYC degradation in vivo. Control or TRIB3-deleted Raji cells were incubated with CHX (10 μg/mL) or CHX plus MG132 (5 μm) for the indicated times. The MYC protein was detected by western blot; GAPDH was used as a loading control. The data are presented as the means ± S.E.M from three independent experiments. d Effect of TRIB3 overexpression on MYC ubiquitination in vivo. HEK 293T cells were transfected with the indicated plasmids for 24 h. Extracts of cells were IP with an anti-HA Ab. Ubiquitinated MYC was detected by immunoblotting. The data are presented as presentative from three independent experiments. e Strategy for screening the potential E3 ligases of MYC. f The interaction of UBE3B and MYC was evaluated by Co-IP assays. Raji cell extracts were IP with rabbit immunoglobulin G (IgG) or an anti-MYC Ab and blotted with an anti-UBE3B Ab. The data are presented as presentative from three independent experiments. g Colocalization of MYC and UBE3B was detected in primary human DLBCL cells (T69) by the Duolink PLA assay. Scale bar, 2 μm. The data are presented as presentative from three independent experiments. h Effect of TRIB3 on MYC degradation mediated by UBE3B. HEK 293T cells were transfected with the indicated plasmids, and 12 h later, incubated with CHX (10 μg/mL) for the indicated times. The data are presented as presentative and the means ± S.E.M from three independent experiments. i Effect of TRIB3 on MYC ubiquitination induced by UBE3B in vivo. HEK 293T cells were transfected with the indicated plasmids for 24 h. Cell extracts were IP with an anti-Flag Ab. Ubiquitinated MYC was detected by immunoblotting. The data are presented as presentative from three independent experiments. j In vitro ubiquitination assays show that purified UBE3B induces polyubiquitination of purified MYC in the presence of UBCH3 (E2), ATP, Uba1 (E1), and biotinylated ubiquitin. The data are presented as presentative from three independent experiments. k In vitro ubiquitination assays show that UBE3B mildly induces polyubiquitination of MYC in the presence of UBCH3 (E2) but not in that of other E2s. The data are presented as presentative from three independent experiments. l Alignment of partial UBE3B sequences (1024–1049 amino acids in human UBE3B) from the indicated species. The cysteine at position 1036 (C1036) of human UBE3B was mutated to alanine (C1036A). In vitro ubiquitination assays showed that the UBE3B C1036A mutant did not induce polyubiquitination of MYC. The data are presented as presentative from three independent experiments. m Mapping UBE3B regions binding to MYC. Left: deletion mutants of UBE3B. Right: HEK 293T cells were cotransfected with the indicated constructs of UBE3B (His tag) and MYC (Flag tag). Cell extracts were IP with an anti-Flag Ab. The data are presented as presentative from three independent experiments. n In vitro assays show that the C terminus of UBE3B (UBE3B-C) mildly induces polyubiquitination of MYC. The data are presented as presentative from three independent experiments. o Pie chart shows all the types and statistics of UBE3B mutations in 89 lymphoma patients. p In vitro ubiquitination assays showed that the UBE3B R346Q mutation decreased the polyubiquitination of MYC compared with the wild-type MYC. The data are presented as presentative from three independent experiments. Source data are provided as a Source Data file. Image collected and cropped by CiteAb from the following open publication (//pubmed.ncbi.nlm.nih.gov/33298911), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Reactivity:

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

Species Glossary

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

Applications:

, , , , , , , , ,

Applications: , , , , , , , , ,

Host:

Mouse Monoclonal

Host: Mouse Monoclonal

Sizes:

Sample Size Available

Sizes: Sample Size Available

Conjugate:

Unconjugated

Available Conjugates:

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

Available Formulations

Formulation	Catalog #	Availability	Price
BSA Free	NB110-67828

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NB100-479

HIF-1 alpha Antibody - BSA Free

(28 Reviews)

(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<0.05, **p<0.01). Also shown are Western blots of nuclear HIF-1alpha at the time of irradiation. Corresponding Western blots of lamin A/C are shown as a loading control. Image collected and cropped by CiteAb from the following publication (//dx.plos.org/10.1371/journal.pone.0111199) licensed under a CC-BY license.

Immunofluorescence of wounded tissue and normal unwounded skin. Representative images for immunofluorescent staining of HIF-1 alpha on healed wounded skin and adjacent normal unwounded skin of wild type, heterozygous K14-Cre/homozygous floxed PHD-2, and heterozygous Col1 alpha2-Cre-ER/homozygous floxed PHD-2 mice at 400x magnification. Image collected and cropped by CiteAb from the following publication (//doi.org/10.1371/journal.pone.0093373), licensed under a CC-BY license.

Staining in normal mouse kidney showing (A) staining with HIF-1 alpha antibody and (B) blocking peptide used at 10-fold excess concentration. Images provided by Dr Yves Heremans. Please see the product review of NBC1-18422 for additional details.

<b>Biological Strategies Validation. </b>Analysis of HIF-1 alpha in mouse hepatocytes (left lane: normoxic and right lane: hypoxic) using HIF-1 alpha antibody. Western blot image submitted by a verified customer review.

Analysis in paraffin section of mouse skin wound using NB100-479 at 1:6000. Image courtesy of product review by Ian Darby of RMIT University.

Immunofluorescence (IF) labeling and confocal microscopy analysis of the distribution of HIF-1alpha (A1-D1) and Abeta (A2-D2) in APPsw cells. DFO treatment significantly increased HIF-1alpha IF (D1) and reduced the Abeta IF (D2) in APPsw cells compared with controls (A1,A2). (A3-D3) DAPI was used to detect the nucleus (blue). (A4-D4) Merged images from the double channels indicated that DFO has an inhibitory effect on Abeta formation, and is involved in HIF-1alpha stabilization in APPsw cells. Fe treatment (100 uM) decreased HIF-1alpha (B1) and increased the Abeta levels (B2). DFO treatment reversed the reduction of HIF-1alpha (C1) and the increase of Abeta (C2) in cultures pretreated with 100 uM Fe. Scale bar = 20 um. Image collected and cropped by CiteAb from the following publication (//journal.frontiersin.org/Article/10.3389/fnagi.2015.00104/abstract) licensed under a CC-BY license.

<b>Biological Strategies Validation. </b>Analysis in A. PC12 (rat) cells, 20% oxygen and B. PC12 (rat) cells, 1% oxygen.

<b>Biological Strategies Validation. </b>Analysis in a hypoxic sample. Lane 1: CoCl2 treated Cos-7 nuclear extract (50 ug, hypoxic). Lane 2: Untreated Cos-7 nuclear extract (50 ug, normoxic). 10 second ECL exposure.

U251 cells exposed to 4 hour hypoxia (0.5% oxygen) incubation. Western blot image submitted by a verified customer review.

Detection of HIF-1 Alpha (green) in RCC4 cells using NB100-479. Nuclei (Blue) were counterstained using Hoechst 33258.

Analysis of HIF-1 alpha in human kidney tumor.

Effects of different routes of CDDP administration on remaining viable tumor tissue. Representative images of tumor tissue (i.v. CDDP, i.a. CDDP) stained for HIF-1alpha (20X). Scale bar corresponds to 100 um. Image collected and cropped by CiteAb from the following publication (//jeccr.biomedcentral.com/articles/10.1186/s13046-016-0392-1), licensed under a CC-BY license.

Hif1alpha expression in preimplantation embryos. Co-staining of OCT4 and Hif1alpha in inner cell mass of blastocyst (top) and negative control images without primary antibodies (bottom). Scale bar: 30um. Image collected and cropped by CiteAb from the following publication (//dx.plos.org/10.1371/journal.pone.0154309) licensed under a CC-BY license.

Simple Western lane view shows lysates of MCF +/- DMOG, BioSpherix MSCs in hypoxic conditions, and BioSpherix MSCs in normoxic conditions loaded at 0.5 mg/ml. A band was detected for HIF-1 alpha at approximately 116 kDa (as indicated) using NB100-479 (1:100 dilution) followed by Anti-Rabbit Secondary Antibody (042-206, ProteinSimple). This experiment was conducted under standard assay conditions, and using the 12-230 kDa separation module (SW-W004). Non-specific interaction with the 230 kDa Simple Western standard seen with this antibody. Image from an internal validation.

<b>Genetic Strategies Validation. </b>Cofilin phosphorylation is HIF-1 alpha dependent.Wt, shC, and the HIF-1 alpha knock down cell clones c1 and c2 cells were lysed after 24 hrs of normoxia (20% O2) or hypoxia (1% O2). Cell extracts were analysed by Western blots. Note that p-cofilin levels are reduced in c2 and c2 cells compared to wt and shC cells at 20% O2 and 1% O2.

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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AF-401-NA

IL-1 beta/IL-1F2 Antibody [Unconjugated]

(3 Reviews)

(520 Publications)

100 μg

AF-401-NA

Western blot shows lysates of THP-1 human acute monocytic leukemia cell line untreated (-) or treated (+) with 200 nM PMA for 24 hours and 10 µg/mL LPS for 4 hours and RAW 264.7 mouse monocyte/macrophage cell line untreated (-) or treated (+) with 10 µg/mL LPS for 24 hours. PVDF membrane was probed with 0.25 µg/mL of Goat Anti-Mouse IL-1 beta /IL-1F2 Antigen Affinity-purified Polyclonal Antibody (Catalog # AF-401-NA) followed by HRP-conjugated Anti-Goat IgG Secondary Antibody (HAF017). A specific band was detected for IL-1 beta /IL-1F2 at approximately 35 kDa (as indicated). This experiment was conducted under reducing conditions and using Immunoblot Buffer Group 1.

IL-1 beta /IL-1F2 was detected in immersion fixed MCF-7 human breast cancer cell line using Goat Anti-Mouse IL-1 beta /IL-1F2 Antigen Affinity-purified Polyclonal Antibody (Catalog # AF-401-NA) at 8 µg/mL for 3 hours at room temperature. Cells were stained using the NorthernLights™ 557-conjugated Anti-Goat IgG Secondary Antibody (red; NL001) and counterstained with DAPI (blue). Specific staining was localized to cytoplasm. View our protocol for Fluorescent ICC Staining of Cells on Coverslips.

IL-1 beta /IL-1F2 was detected in perfusion fixed frozen sections of mouse thymus using Goat Anti-Mouse IL-1 beta /IL-1F2 Antigen Affinity-purified Polyclonal Antibody (Catalog # AF-401-NA) at 15 µg/mL overnight at 4 °C. Tissue was stained using the Anti-Goat HRP-DAB Cell & Tissue Staining Kit (brown; CTS008) and counterstained with hematoxylin (blue). View our protocol for Chromogenic IHC Staining of Frozen Tissue Sections.

Simple Western lane view shows lysates of RAW 264.7 mouse monocyte/macrophage cell line untreated (-) or treated (+) with 10 µg/mL LPS for 24 hours, loaded at 0.5 mg/mL. A specific band was detected for IL‑1 beta /IL‑1F2 at approximately 40 kDa (as indicated) using 2.5 µg/mL of Goat Anti-Mouse IL‑1 beta /IL‑1F2 Antigen Affinity-purified Polyclonal Antibody (Catalog # AF-401-NA) followed by 1:50 dilution of HRP-conjugated Anti-Goat IgG Secondary Antibody (Catalog # HAF109). This experiment was conducted under reducing conditions and using the 12-230 kDa separation system.

Recombinant MouseIL‑1 beta /IL‑1F2 (Catalog # 401-ML) stimulates proliferation in the the D10.G4.1 mouse helper T cell line in a dose-dependent manner (orange line) as measured by Resazurin (Catalog # AR002). Proliferation elicited by Recombinant Mouse IL‑1 beta /IL‑1F2 (50 pg/mL) is neutralized (green line) by increasing concentrations of Goat Anti-Mouse IL-1 beta /IL-1F2 Antigen Affinity-purified Polyclonal Antibody (Catalog # AF-401-NA). The ND50 is typically ≤0.25 µg/mL.

Quercetin inhibits auto-reactive NLRP3 inflammasome.(A) IL-1 beta concentrations in Nlrp3A350V/A350V and WT BMDM culture supernatants primed with LPS (500 ng/ml) for 90 min and treated with quercetin 1 h after LPS. (B) Supernatants of BMDM were analyzed by anti-ASC, anti-Caspase-1 immunoblotting. Lysates of BMDM were analyzed by anti-IL-1 beta and anti-beta -actin immunoblotting. Data shown are representative of two or more experiments (means ± SD) ***p VC006). Before incubation with the primary antibody, tissue was subjected to heat-induced epitope retrieval using VisUCyte Antigen Retrieval Reagent-Basic (Catalog # VCTS021). Tissue was stained using DAB (brown) and counterstained with hematoxylin (blue). Specific staining was localized to cytoplasm in epithelial cells. View our protocol for IHC Staining with VisUCyte HRP Polymer Detection Reagents.

GRA7-I-induced inflammasome activation was required for antimicrobial activity in MTB-infected macrophages.(A and B) BMDMs from PKC alpha +/+ and PKC alpha -/- mice (A) and BMDMs were transduced with lentivirus-shRNA-NS or lentivirus-shRNA-ASC for 2 days (B) infected with MTB (MOI = 1) for 4 h and then stimulated with rGRA7 (1, 5, 10 μg/ml) and its mutants for 18 h. IB analysis for IL-1 beta p17, IL-18 p18, or caspase-1 p10 in supernatants (SN), ASC, NLRP3, pro-IL-1 beta , pro-IL-18, or pro-caspase-1 in whole-cell lysates (WCL). Actin was used as a loading control. (C and D) Intracellular survival of MTB was assessed by CFU assay. BMDMs were infected with MTB for 4 h, followed by treatment with rGRA7, and then lysed to determine intracellular bacterial loads. The data are representative of five independent experiments with similar results (A and B). Data shown are the mean ± SD of five experiments (C and D). Significant differences (*P p>0.001,***p<0.001, ns = not significant ) as determined by one way ANOVA with Tukey's post test. Image collected and cropped by CiteAb from the following publication (//dx.plos.org/10.1371/journal.pone.0040722), licensed under a CC-BY license. Not internally tested by R&D Systems.

Effects of IL-1 beta neutralization on the accumulation of MDSCs in lungs.(A) Immunofluorescent photos showed that Gr-1+ (green)/CD11b+ (red) cells indicate MDSCs and increased in lungs of DJ-1 KO mice. Treatment with anti-IL-1 beta neutralizing antibody antagonized the accumulation of MDSCs. DAPI was used to stain nuclei. (B) Bar chart showed the summarized results of MDSCs in lungs of WT and DJ-1 KO mice. Note that the neutralizing antibody significantly reduced the number of MDSCs accumulated in lungs. n = 9 for each group, * p < 0.05 compared with control IgG-treated WT mice, # p<0.05 compared with respective control IgG in WT and DJ-1 KO mice. Image collected and cropped by CiteAb from the following publication (//dx.plos.org/10.1371/journal.pone.0115827), licensed under a CC-BY license. Not internally tested by R&D Systems.

Pro-IL-1 beta degradation is induced by the autophagy inhibitors chloroquine and bafilomycin A, and by the proteasome activator betulinic acid. (A) Chloroquine selectively attenuates IL-1 beta release from cardiac fibroblasts. The cells were incubated with LPS and/or 20 μM chloroquine phosphate as indicated for 18 h, then activated with 3 mM ATP for 60 min. IL-1 beta , TNF, MIP-2, and IL-6 were quantified in the conditioned media (n = 3). (B,C) Cardiac fibroblasts were incubated with LPS for 18 h with or without 20 μM chloroquine phosphate and pro-IL-1 beta quantified with western blot (n = 3). (D) Pro-IL-1 beta mRNA expression was quantified with qPCR (n = 3). (E,F) Cardiac fibroblasts were incubated with LPS for 20 h with or without 10% FCS or 20 μM chloroquine phosphate as indicated. The amount of total ubiquitinated protein was quantified with western blot (n = 4). (G,H) Cardiac fibroblasts were incubated with 10% FCS with or without 10 ng/mL LPS and/or 100 nM bafilomycin A1 for 20 h. Pro-IL-1 beta was quantified with western blot. (I,J) Cardiac fibroblasts were incubated with 10% FCS with or without 10 ng/mL LPS and/or 20 μM betulinic acid as indicated for 20 h. Pro-IL-1 beta was quantified with western blot. All columns are mean with SEM. *p < 0.05, **p < 0.01 (paired student t-test). Image collected and cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/31244838), licensed under a CC-BY license. Not internally tested by R&D Systems.

Pro-IL-1 beta maturation is regulated by POH1-mediated deubiquitination. a, b HEK293T cells were transfected with plasmids encoding Flag-caspase-1, Vsv-ASC, His-pro-IL-1 beta (WT) or His-pro-IL-1 beta -K133R with different amounts (125, 250, 500 ng) of plasmid encoding Flag-POH1 and 500 ng of control plasmid, the secretion of a IL-1 beta in the supernatants was quantified by ELISA 36 h after transfection; b the cell lysates were IB with the indicated antibodies. c Mutants of murine pro-IL-1 beta with replacement of various lysine residues. d-f HEK293T cells were transfected with Flag-POH1, V5-caspase-1, V5-ASC, His-pro-IL-1 beta (WT) or its mutants, along d with or e-f without HA-tagged K63-only Ub, then d cells were subjected to IP with an anti-His antibody and IB with the indicated antibodies. e The IL-1 beta levels in supernatants were measured by ELISA; or f the cell lysates were collected and IB with the indicated antibodies. g Proposed function of POH1 as a negative regulator in mediating IL-1 beta processing. POH1, upregulated by TLR3/4 activation, interacts with and deubiquitinates pro-IL-1 beta , resulting in restriction of IL-1 beta cleavage and secretion. Similar results were obtained from three independent experiments. The results represent the mean ± s.d. of three independent sets of experiments. **p < 0.01, ***p < 0.001 (one-way ANOVA with Tukey’s post-hoc test) Image collected and cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/30315153), licensed under a CC-BY license. Not internally tested by R&D Systems.

Serum starvation selectively induces pro-IL-1 beta degradation in cardiac fibroblasts. (A) Cells were incubated with 10 ng/mL LPS for 5 or 20 h with or without 10% FCS and western blot analysis of pro-IL-1 beta and NLRP3 performed. Blots are representative of 3 independent experiments. (B) Protein bands were quantified and the ratios to beta -actin normalized to mean control = 1 (n = 3). (C,D) Cardiac fibroblasts were incubated with or without 10% FCS and primed with LPS for 20 h. Western blot analysis of complex II (mitochondrial mass marker) and NLRP3- inflammasome protein components (NEK7, NLRP3, ASC, pro-caspase-1, and pro-IL-1 beta ) were performed. Bands were quantified and the ratios to beta -actin normalized to mean control = 1 (n = 4, for ASC n = 10). (E,F) Cardiac fibroblasts (120,000 cells per well seeded in 6 well plates) were incubated with 10 ng/mL LPS for 20 h, with or without 10% FCS, then stained with 500 nM MitoTracker Deep Red for 45 min before MitoTracker fluorescence intensity was quantified with flow cytometry analysis (633 nm laser). All cells were analyzed and all gated cells included in the analysis. The presented dot plots (E) show the gate in red and are representative for 9 biological repeats. Median MitoTracker intensity with and without 10% FCS are shown (F). All columns are mean with SEM. *p < 0.05,**p < 0.01 (paired student t-test). Image collected and cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/31244838), licensed under a CC-BY license. Not internally tested by R&D Systems.

Differential effects of IL-1 beta neutralization on melanoma lung nodules in WT and DJ-1 KO mice.(A) IL-1 beta antibody (10μg) or control IgG was intravenously injected into mice once. The melanoma cells (6×104) were intravenously injected 30 minutes later. The mice were sacrificed three weeks later. Bar chart showed that the anti-IL-1 beta neutralizing antibody can effectively lower serum IL-1 beta levels as compared with control IgG in both WT and DJ-1 KO mice. Note that serum IL-1 beta levels were higher in DJ-1 KO mice than those in WT mice. (B) Inhibition of IL-1 beta neutralization on lung nodules formation in DJ-1 KO mice. Upper panels: melanoma nodules in lungs of WT (top) and KO (bottom) mice treated with control IgG (left) or neutralizing anti-IL-1 beta antibody (right). For each experimental condition, the gross and microscopic morphologies of tumor nodules are indicated by arrows, and are shown at left and right sites, respectively. Scale bars: 0.5 mm for lung photographs and 0.5 mm for H&E staining. Lower panel: Bar chart showed the summarized results of lung nodule numbers in WT and DJ-1 KO mice. Note that IL-1 beta neutralization enhanced and suppressed melanoma nodules formation in WT and DJ-1 KO mice, respectively. Data are presented as mean ± S.E.M. n = 5 for each group, * p < 0.05 compared with control IgG-treated WT mice, # p<0.05 compared with respective control IgG in WT and DJ-1 KO mice. Image collected and cropped by CiteAb from the following publication (//dx.plos.org/10.1371/journal.pone.0115827), licensed under a CC-BY license. Not internally tested by R&D Systems.

Inflammasome assembly and mature IL-1 beta release in the spinal cord after sciatic nerve axotomy. a Speck-like costaining of NLRP3 and ASC in injured neurons 3 days after the injury (arrows). b Representative WB images of mature IL-1 beta and beta -actin proteins 3 days after unilateral axotomy of sciatic nerve. c Quantification of active IL-1 beta protein expression. N = 3 animals/group. The graph shows values normalized to beta -actin levels and relative to contralateral side. Mean values are shown on bars. Bars represent average ± SEM. **p < 0.01 (ANOVA with Bonferroni post hoc, ax. + vehicle vs. contralateral side). #p < 0.05, ##p < 0.01 (ANOVA with Bonferroni post hoc, compared to ax. + vehicle). Ax. axotomy Image collected and cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/35305649), licensed under a CC-BY license. Not internally tested by R&D Systems.

$GRA7-I activated inflammasomes in an ASC-binding dependent manner.(A) Bacterially purified 6xHis-GRA7-I and its mutants were analyzed by Coomassie blue staining (left) or IB with alpha His (right). (B) BMDMs from PKC alpha +/+ and PKC alpha -/- (left) or BMDMs were transduced with lentivirus-shRNA-NS or lentivirus-shRNA-ASC (MOI = 100) with polybrene (8 μg/mL) (right) for 2 days, the cells were stimulated with rGRA7 (5 μg/ml) and its mutants for the indicated times and culture supernatants were harvested and analyzed for cytokine ELISA for IL-1 beta and IL-18. Data shown are the means ± SD of five experiments. Significant differences (*P < 0.05; ***P < 0.001) compared with PKC alpha +/+ or shRNA-NS. (C) IB analysis for IL-1 beta p17, IL-18 p18, or caspase-1 p10 in supernatants (SN), ASC, NLRP3, pro-IL-1 beta , pro-IL-18, or pro-caspase-1 in whole-cell lysates (WCL). Actin was used as a loading control. (D) IB analysis of lysates of BMDMs as in B and C solubilized with Triton X-100–containing buffer, followed by cross-linkage of insoluble fractions with disuccinimidyl suberate to capture ASC oligomers and analysis of those fractions (I + DSS) and soluble fractions (S) with antibody to ASC. Actin was used as a loading control. (E) Fluorescence confocal images showing formation of speck-like ASC pyroptosomes in BMDMs from PKC alpha +/+ and PKC alpha -/- mice were stimulated with rGRA7 and its mutants for 18 h, fixed, immunostained with antibodies for ASC (Alexa 488). Scale bar, 10 μm. The data are representative of five independent experiments with similar results (A and C-E). Image collected and cropped by CiteAb from the following publication (//dx.plos.org/10.1371/journal.ppat.1006126), licensed under a CC-BY license. Not internally tested by R&D Systems.$

The B6 variant of NLRP1B is not cleaved by LF but can form an inflammasome in response to proteolysis.(A) The amino acid sequence of the first 244 residues of NLRP1BB6 was aligned to the homologous sequence of NLRP1B129. The arrow above the alignment indicates the LF-cleavage site in NLRP1B129. Asterisks indicate sites of amino acid identity. (B) For detection of NLRP1B expression, 293T cells were transfected with the indicated amounts of and empty vector (V) or plasmids encoding GFP-HA-NLRP1B129 or GFP-HA-NLRP1BB6 (construct schematics and the predicted molecular weight of each protein is depicted in the upper panel). Cells were treated overnight with anthrax lethal toxin protein (LeTx, 1μg/ml) 24h post-transfection, and then lysates were analyzed by immunoblotting (IB) with the indicated antibodies. For NLRP1 expression and cleavage, CASP1 and IL1B were omitted to prevent cell death and resulting apparent differences of expression. For blots probed with anti-HA (NLRP1B), the lysates were not boiled prior to loading to prevent aggregation and smearing of full-length and FIIND-processed NLRP1B on the immunoblot. To visualize the N-terminal processed form of NLRP1B, the lysates were boiled and resolved on a separate gel. (C) 293T Cells were transfected with the same amount of titrated NLRP1B encoding plasmid and treated as in B, but transfections also included plasmids encoding mouse CASP1 (200ng), IL-1 beta (200ng) and 200ng of empty vector. (D) For detection of NLRP1B expression, 293T cells were transfected for 36h with 250ng of plasmids encoding WT 129 or B6 NLRP1B or mutants engineered to express the TEV-protease site. Each plasmid was co-transfected with either 100ng of empty vector (V) or plasmids encoding TEV-protease (pTEV) or lethal factor protease (pLF), supplemented with 300ng empty vector (pMSCV). Cells were not treated with LeTx and lysates were analyzed by immunoblotting as in (B). (E) Cells were treated as in C, but with 8ng of plasmids encoding NLRP1B, along with 200ng of plasmids encoding mCASP-1 and mIL-1 beta , and 200ng empty vector to normalize plasmid quantities. Lower quantities of NLRP1B were transfected as compared to panel D so to avoid spontaneous NLPR1B activation. For panels B-E, data shown are representative of at least three similar experiments. Image collected and cropped by CiteAb from the following publication (//dx.plos.org/10.1371/journal.ppat.1006052), licensed under a CC-BY license. Not internally tested by R&D Systems.

SHP inhibits the signal 2 activation of NLRP3 inflammasome.(a,b) LPS-primed BMDMs were treated with fenofibrate (10, 20, 50 μM for 4 h), or (c,d) LPS-primed THP-1 cells were treated with recombinant macrophage-stimulating protein (MSP 10, 50, 100 ng ml−1 for 4 h), and then activated with ATP (5 mM) for 30 min, followed by IB analysis of IL-1 beta p17 or caspase-1 p10 in supernatants (SN), pro-IL-1 beta or pro-caspase-1 in whole-cell lysates (WCL), with actin as a loading control. Data are representative of three independent experiments (a,c). Supernatants were collected and subjected to ELISA for IL-1 beta , IL-18, TNF-alpha and IL-8. Data are the means±s.d. of values from four independent experiments (b,d). *P<0.05; **P<0.01; ***P<0.001, compared with control (two-tailed Student’s t-test). FF, fenofibrate. Image collected and cropped by CiteAb from the following publication (//www.nature.com/articles/ncomms7115), licensed under a CC-BY license. Not internally tested by R&D Systems.

NLRP1A is activated by N-terminal proteolysis.(A) The amino acid sequence of the first 244 residues of NLRP1AB6 was aligned to NLRP1B129 and NLRP1BB6. The arrow above the alignment indicates the LF-cleavage site in NLRP1B129. (B) 293T cells were transfected for 36h with 200ng empty vector (V) or plasmids pcDNA3.1-HA-NLRP1B129 or -NLRP1AB6-MYC (CMV promoter) along with 200ng mCASP1 and 200ng mIL-1 beta expression vectors. Cells were treated overnight with anthrax lethal toxin (LeTx, 1μg/ml) 24h post-transfection, and then lysates were analyzed by immunoblotting (IB) with the indicated antibodies. (C) 293T cells were transfected for 36h with plasmids encoding 400ng GFP-HA-NLRP1B129 or GFP-HA-NLRP1AB6 (under the control of the LTR promoter in pMSCV) or with mutants engineered to contain an N-terminal TEV protease site. Cells were also co-transfected with 100ng empty vector (V) or plasmids encoding TEV-protease (pTEV) or lethal factor protease (pLF), plus 300ng empty pMSCV. (D) To assess IL-1 beta cleavage, cells were transfected as in C, but with 8ng of GFP-HA-NLRP1B and co-transfected with vectors encoding 200ng mCASP1 and 200ng mIL-1 beta . For panels B-D, data shown are representative of at least three similar experiments. Image collected and cropped by CiteAb from the following publication (//dx.plos.org/10.1371/journal.ppat.1006052), licensed under a CC-BY license. Not internally tested by R&D Systems.

POH1 inhibits inflammasome-induced pro-IL-1 beta processing. a-c BMDMs from Poh1 delta /+ and Poh1 delta / delta mice were primed with LPS for 12–14 h and then stimulated with ATP (0.5 h), nigericin (Nig, 0.5 h), poly(dA:dT) (5 h) or flagellin (5 h) as indicated, a IL-1 beta , b IL-6 and c TNF alpha levels in supernatants were analysed by ELISA. d-f BMDMs lentivirally transduced with control (Vector) or POH1 were stimulated as in a, d IL-1 beta , e IL-6 and f TNF alpha levels in supernatants were analysed by ELISA. g, h BMDMs g derived from Poh1 delta /+ and Poh1 delta / delta mice or h infected by indicated lentiviruses were treated as in a, the cell lysates (CL) and supernatants (SN) were collected and immunoblotted with the indicated antibodies. Similar results were obtained from three independent experiments. The results represent the mean ± s.d. of three independent sets of experiments. **p < 0.01, ***p < 0.001 (two-tailed Student’s t-test) Image collected and cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/30315153), licensed under a CC-BY license. Not internally tested by R&D Systems.

Quercetin inhibits NLRP3 and AIM2 Inflammasomes.LPS (500 ng/ml)-primed BMDM were treated with the indicated flavonoids and concentrations or vehicle (DMSO 0.01%), then stimulated with (A) 5 mM ATP (B) 10 μM Nigericin (C) 130 μg/ml Alum or (D) 400 ng/ml Poly (dA:dT) 30 minutes after treatment. (A–D) IL-1 beta and (E) TNF-alpha concentration in the culture supernatant were measured by ELISA. (B,F,G) LPS-primed BMDM were stimulated with Nigericin treated with quercetin 30 minutes before secondary stimulation. Supernatants and Lysate of BMDM were analyzed by immunoblotting. Cells were stained with FLICA and Caspase-1 activation was determined by FLICA positivity. Data shown are representative of two or more independent experiments (means ± SD) *p < 0.05, **p < 0.01, ***p < 0.001. Image collected and cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/28148962), licensed under a CC-BY license. Not internally tested by R&D Systems.

P. gingivalis and its OMVs differentially induce inflammasome signaling and pyroptosis in human macrophages. MDM were infected as before (2 h at MOI of 25:1, see Materials and Methods) with viable P. gingivalis (Pg), heat-killed-Pg (HK-Pg), OMVs, or heat-inactivated-OMVs (HI-OMVs) and (A) the activation of inflammasome components in the lysates was measured after 24 h by Western blot; beta -actin serves as a loading control throughout. Western blot data are representative of at least three independent experiments. (B) Production of IL-1 beta and IL-18 (by ELISA) from MDM was measured in the supernatant at 24 h. Data are mean ± SD from four independent experiments. Cell viability was determined by measuring extracellular LDH release and by 7-AAD exclusion (by flow-cytometry) at 24 h. 7-AAD is a membrane impermeant dye that is excluded from viable cells. The percent of 7-AAD positive (±SD) cells from three independent experiments was calculated relative to the untreated control. MDM treated with H2O2 (1 mM for 60 min) were included as positive control for 7-AAD. A representative histogram is shown. Cells were also treated where indicated with OMVs in the presence of KYT-1 and KYT-36 (10 μM of each). *p < 0.05: n.s, not significant. Image collected and cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/28824884), licensed under a CC-BY license. Not internally tested by R&D Systems.

P. gingivalis and its OMVs differentially induce inflammasome signaling and pyroptosis in murine macrophages. BMM were infected as before (2 h at MOI of 25:1, see Materials and Methods) with viable P. gingivalis (Pg), heat-killed-Pg (HK-Pg), OMVs, or heat-inactivated-OMVs (HI-OMVs) and (A) the activation of inflammasome components in the lysates [or supernatants (sup) where indicated] measured after 24 h by Western blot; beta -actin serves as a loading control throughout. Western blot data are representative of at least three independent experiments. (B) Production of IL-1 beta and IL-18 (by ELISA) from BMM was measured in the supernatant at 24 h. Data are mean ± SD from four independent experiments. Cell viability was determined by measuring extracellular LDH release and by 7-AAD exclusion (by flow-cytometry) at 24 h. 7-AAD is a membrane impermeant dye that is excluded from viable cells. The percent of 7-AAD positive (±SD) cells from three independent experiments was calculated relative to the untreated control. BMM treated with H2O2 (1 mM for 60 min) were included as positive control for 7-AAD. A representative histogram is shown. Cells were also treated where indicated with OMVs in the presence of KYT-1 and KYT-36 (10 μM of each). *p < 0.05. Image collected and cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/28824884), licensed under a CC-BY license. Not internally tested by R&D Systems.

Caspase-1 but not gasdermin-D processing depends on ASC oligomerization.(a) Western blot analysis for cleaved caspase-1 p20, IL-1 beta p17, and HMGB-1 in cell supernatants (SN) and pro-caspase-1, pro-IL-1 beta and HMGB-1 in cell lysates (lys) of LPS-primed immortalized Asc−/− BMDMs or Asc−/− BMDMs expressing ASCFL, ASCY59A or ASCE80R 3 h after poly(dA:dT) transfection (1 μg ml−1). (b) Release of LDH from LPS-primed immortalized Asc−/− BMDMs or Asc−/− BMDMs expressing ASCFL, ASCY59A or ASCE80R, or derived Casp1 knockouts 3 h after poly(dA:dT) transfection (1 μg ml−1). (c) Western blot analysis for processing of full-length gasdermin-D (GSDMDFL) into the active N-terminal fragment (GSDMDN-term) in combined lysates and supernatants (lys + SN) of LPS-primed immortalized Asc−/− BMDMs expressing ASCFL, ASCY59A or ASCE80R, or derived Casp1 knockouts 3 h after poly(dA:dT) transfection (1 μg ml−1). Arrowhead, gasdermin-DNterm p30; *a cross-reacting band. (d) Release of LDH from LPS-primed primary C57BL/6 WT (WT), Casp1−/−/Casp11−/− or Asc−/− BMDMs infected with S. Typhimurium (multiplicity of infection (MOI)=10, 1 h). (e) Western blot analysis for processing of full-length gasdermin-D (GSDMDFL) into the active N-terminal fragment (GSDMDN-term) in combined lysates and supernatants (lys+SN) of LPS-primed primary C57BL/6 WT (WT), Casp1−/−/Casp11−/− or Asc−/− BMDMs infected with S. Typhimurium (MOI=10, 1 h) or left uninfected. Arrowhead, gasdermin-DNterm p30; *a cross-reacting band; ** a S. Typhimurium-specific cross-reactive band. See also Supplementary Figs 8 and 9. Image collected and cropped by CiteAb from the following publication (//www.nature.com/articles/ncomms11929), licensed under a CC-BY license. Not internally tested by R&D Systems.

Nimbidiol treatment mitigated elevated pro-inflammatory cytokine and chemokine in the diabetic kidney. Representative images from (A) Semi-quantitative RT-PCR analyses showing gene expression and (B) western blot analyses showing protein expression of TNF-alpha , IL-1 beta and MCP-1 in the kidney. The bar diagrams represent the fold change from WT + Saline. Data are mean ± SD (n = 6/group). *p < 0.05 versus WT + Saline, WT + Nimbidiol and Akita + Nimbidiol, †p < 0.05 versus Akita + Saline. Image collected and cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/36522378), licensed under a CC-BY license. Not internally tested by R&D Systems.

Reactivity:

Mu

Species Glossary

Reactivity: Mu

Applications:

, , , ,

Applications: , , , ,

Host:

Goat Polyclonal

Host: Goat Polyclonal

Sizes:

Sample Size Available

Sizes: Sample Size Available

Conjugate:

Unconjugated

Available Conjugates:

Conjugate	Catalog #	Availability	Size	Price
Biotin	BAF401

Available Formulations

Formulation	Catalog #	Availability	Price
Carrier Free	AF-401-SP

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AF3628

CD31/PECAM-1 Antibody [Unconjugated]

(6 Reviews)

(477 Publications)

100 μg

AF3628

Western blot shows lysates of bEnd.3 mouse endothelioma cell line. PVDF membrane was probed with 0.5 µg/mL of Goat Anti-Human/Mouse/Rat CD31/PECAM-1 Antigen Affinity-purified Polyclonal Antibody (Catalog # AF3628) followed by HRP-conjugated Anti-Goat IgG Secondary Antibody (HAF017). A specific band was detected for CD31/PECAM-1 at approximately 130 kDa (as indicated). This experiment was conducted under reducing conditions and using Immunoblot Buffer Group 1.

Rat splenocytes were stained with Goat Anti-Human/Mouse/Rat CD31/PECAM-1 Antigen Affinity-purified Polyclonal Antibody (Catalog # AF3628, filled histogram) or isotype control antibody (AB-108-C, open histogram), followed by Allophycocyanin-conjugated Anti-Goat IgG Secondary Antibody (F0108).

CD31/PECAM-1 was detected in immersion fixed bEnd.3 mouse endothelioma cell line using Goat Anti-Human/Mouse/Rat CD31/PECAM-1 Antigen Affinity-purified Polyclonal Antibody (Catalog # AF3628) at 10 µg/mL for 3 hours at room temperature. Cells were stained using the NorthernLights™ 493-conjugated Anti-Goat IgG Secondary Antibody (green; NL003) and counterstained with DAPI (blue). Specific staining was localized to cell membrane. View our protocol for Fluorescent ICC Staining of Cells on Coverslips.

CD31/PECAM-1 was detected in immersion fixed frozen sections of mouse embryo (E13.5) using Goat Anti-Human/Mouse/Rat CD31/PECAM-1 Antigen Affinity-purified Polyclonal Antibody (Catalog # AF3628) at 10 µg/mL overnight at 4 °C. Tissue was stained using the NorthernLights™ 557-conjugated Anti-Goat IgG Secondary Antibody (yellow; NL001) and counterstained with DAPI (blue). Specific staining was localized to developing endothelium. View our protocol for Fluorescent IHC Staining of Frozen Tissue Sections.

CD31/PECAM-1 was detected in immersion fixed frozen sections of mouse embryo (14 d.p.c.) using Goat Anti-Human/Mouse/Rat CD31/PECAM-1 Antigen Affinity-purified Polyclonal Antibody (Catalog # AF3628) at 10 µg/mL for 1 hour at room temperature followed by incubation with the Anti-Goat IgG VisUCyte™ HRP Polymer Antibody (VC004). Tissue was stained using DAB (brown) and counterstained with hematoxylin (blue). Specific staining was localized to developing guts. View our protocol for IHC Staining with VisUCyte HRP Polymer Detection Reagents.

CD31/PECAM‑1 was detected in immersion fixed paraffin-embedded sections of rat heart using Goat Anti-Human/Mouse/Rat CD31/PECAM‑1 Antigen Affinity-purified Polyclonal Antibody (Catalog # AF3628) at 3 µg/mL for 1 hour at room temperature followed by incubation with the Anti-Mouse IgG VisUCyte™ HRP Polymer Antibody (VC001). Before incubation with the primary antibody, tissue was subjected to heat-induced epitope retrieval using Antigen Retrieval Reagent-Basic (CTS013). Tissue was stained using DAB (brown) and counterstained with hematoxylin (blue). Specific staining was localized to endothelial cells in vasculature.

CD31/PECAM‑1 was detected in immersion fixed paraffin-embedded sections of Human Placenta using Goat Anti-Human/Mouse/Rat CD31/PECAM‑1 Antigen Affinity-purified Polyclonal Antibody (Catalog # AF3628) at 15 µg/mL for 1 hour at room temperature followed by incubation with the Anti-Goat IgG VisUCyte™ HRP Polymer Antibody (Catalog # VC004). Before incubation with the primary antibody, tissue was subjected to heat-induced epitope retrieval using VisUCyte Antigen Retrieval Reagent-Basic (Catalog # VCTS021). Tissue was stained using DAB (brown) and counterstained with hematoxylin (blue). Specific staining was localized to endothelial cells in chorionic villi. View our protocol for IHC Staining with VisUCyte HRP Polymer Detection Reagents.

HUVEC cells were stained with Goat Anti-Human/Mouse/Rat CD31/PECAM‑1 Antigen Affinity-purified Polyclonal Antibody (Catalog # AF3628, filled histogram) or isotype control antibody (Catalog # AB-108-C, open histogram), followed by Phycoerythrin-conjugated Anti-Goat IgG Secondary Antibody (Catalog # F0107). View our protocol for Staining Membrane-associated Proteins.

Western blot shows lysates of U937 human histiocytic lymphoma cell line. PVDF membrane was probed with 0.5 µg/ml of Goat Anti-Human/Mouse/Rat CD31/PECAM‑1 Antigen Affinity-purified Polyclonal Antibody (AF3628) followed by HRP-conjugated Anti-Goat IgG Secondary Antibody (Catalog # HAF017). A specific band was detected for CD31/PECAM‑1 at approximately 130 kDa (as indicated). This experiment was conducted under reducing conditions and using Western Blot Buffer Group 1.

Representative image of immunostaining corresponding to the glucose transporter protein SLC2A1 (GLUT1) in 30 week-old Ins2AKITA and WT cerebral cortex. Mural cells (ANPEP, green), glucose transporter 1 (GLUT1, red), endothelium (PECAM1, cyan). n = 2, scale bar 50 µm. Image collected and cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/30498224), licensed under a CC-BY license. Not internally tested by R&D Systems.

AcSDKP suppresses TGF beta /smad signaling and EndMT through the FGFR1/FRS2 pathway. (a) HMVECs were treated with N-FGFR1 for 48 h, and the FGFR1, TGF beta R1 and TGF beta R2 protein levels were analyzed by western blot. (b) HMVECs were treated with TGF beta 2 in the presence or absence of N-FGFR1 for 15 min with or without AcSDKP preincubation. The p-smad3 and TGF beta R1 protein levels were analyzed by western blot. Densitometric analysis of the p-smad3/smad3 and TGF beta R1/ beta -actin levels (n=3) in each group was performed. (c) HMVECs were incubated with either N-FGFR1 in the presence or absence of TGF beta 2 for 48 h with or without preincubation with AcSDKP for 2 h or with N-FGFR1 in the presence or absence of TGF beta 2 for 48 h with or without 24 h of incubation with FGF2 (50 ng/ml). The CD31, SM22 alpha , FSP1 and alpha -SMA protein levels were analyzed by western blot. (d) HMVECs were transfected with FRS2 siRNA (100 nM) for 48 h with or without AcSDKP preincubation. The VE-cadherin, FSP1, vimentin, SM22 alpha and p-smad3 levels were analyzed by western blot. (e) HMVECs were treated with N-FGFR1 for 48 h or 15 min in the presence or absence of N-TGF beta (1, 2, 3) (1.0 μg/ml). The CD31, VE-cadherin, SM22 alpha , FSP1, TGF beta R1, TGF beta R2 and p-smad3 levels were analyzed by western blot Image collected and cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/28771231), licensed under a CC-BY license. Not internally tested by R&D Systems.

AcSDKP inhibits TGF beta /smad signaling and EndMT and restores the FGFR1 and P-MAP4K4 levels in diabetic hearts. (a) Immunofluorescence microscopy analysis of CD31/FGFR1 and CD31/P-MAP4K4 in the heart tissues from each group of mice. The scale bar is 60 μm in each panel. The CD31 and FGFR1 double-labeled cells and the CD31 and P-MAP4K4 double-labeled cells in each visual field were assessed by fluorescence microscopy and quantified. For each section, images from six different fields of view at × 400 magnification were evaluated. (b and c) Immunofluorescence microscopy analysis of CD31/ alpha -SMA, VE-cadherin /SM22 alpha and CD31/p-smad3 expression levels in the heart tissues from each group of mice. The scale bar is 60 μm in each panel. The CD31 and alpha -SMA double-labeled cells, the VE-cadherin and SM22 alpha double-labeled cells and the CD31 and p-smad3 double-labeled cells in each visual field were analyzed by fluorescence microscopy and quantified. For each section, images from six different fields of view at × 400 magnification were evaluated. Four mice from each group were analyzed. (d) Western blot analysis of the FGFR1, P-MAP4K4, TGF beta 1, TGF beta 2 and TGF beta 3 levels in cardiac tissues. A representative blot from four independent experiments was shown. The densitometric analysis of western blot data was presented (n=4). The diabetic mice are abbreviated as DM in the figure Image collected and cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/28771231), licensed under a CC-BY license. Not internally tested by R&D Systems.

Diminished permeability of liver sinusoids in Plvap-deficient mice.Neither by immunohistochemistry with antibodies against CD31 (A) nor by light microscopy of 1 µm semi-thin sections (B, Richardson's stain) obvious differences are detected with regards to the overall orientation and the density of liver sinusoids between 3-week-old Plvap-/- mice and wild-type littermates. Sinusoids of Plvap-deficient mice show a higher number of macrophages in their lumen (white arrows) and focal areas with accumulations of mononuclear cells in Disse's space (black arrows). Lower panels in B show higher magnifications. C, After perfusion of a wild-type animal with FITC-dextran, a strong FITC-signal (green) throughout the liver is detected. Immunolabeling with CD31 (red) suggests that FITC-dextran molecules have accumulated in the space of Disse. In contrast, in the Plvap-deficient littermate, the signal for FITC-dextran is much weaker and barely detectable. Nuclear DNA is labeled with DAPI (blue). Image collected and cropped by CiteAb from the following publication (//dx.plos.org/10.1371/journal.pone.0115005), licensed under a CC-BY license. Not internally tested by R&D Systems.

Blood-brain barrier permeability measurements in male Ins2AKITA and WT littermate controls. (a) Representative stereomicroscope fluorescence images of brains showing 1 kDa Alexa Fluor 555 cadaverine permeability in Ins2AKITA and WT after 2 h of dye circulation (n = 2). (b) Representative confocal images of coronal sections of 1 kDa Alexa Fluor 555 cadaverine injected mouse brains. ANPEP positive mural cells, green; PECAM1 positive vasculature, white. No Alexa Fluor 555 cadaverine leakage into brain parenchyma was observed either in Ins2AKITA or WT mice (n = 2, scale bar 30 μm). (c) Quantification of 1 kDa Alexa Fluor 555 cadaverine permeability in 26.5–32 week-old Ins2AKITA and WT mice after 2 h circulation (n = > 8, 3 independent experiments). y-axis shows the fold change in relative fluorescence units (RFU) per gram of brain tissue in relation to WT. (d) Quantification of 1 kDa Alexa Fluor 488 cadaverine permeability in 38 week-old Ins2AKITA and WT mice after 2 h circulation (n = 3). y-axis shows the fold change in relative fluorescence units (RFU) per gram of brain tissue in relation to WT. (e) Evans Blue dye permeability in 30 week-old Ins2AKITA and WT littermate control mice after overnight circulation (n = > 2). y-axis shows optical density (OD) at 620 nm per gram of tissue. PdgfbRet/Ret served as positive control for tracer leakage into the brain parenchyma. n.s., not significant, student’s t test. Data is presented as mean ± SEM. Image collected and cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/30498224), licensed under a CC-BY license. Not internally tested by R&D Systems.

CL and low temperature lead to WAT browning, angiogenesis, and an increased number of myofibroblast-like cells. a, b Histological analysis of adipocyte morphology (H&E), adipocytes (PERI), mitochondria (COX4), uncoupling protein 1 (UCP1), blood vessels (CD31), and myofibroblast-like cells ( alpha SMA) in a 5-day CL-316243-treated scWAT and visWAT compared to vehicle-treated control. b Two-week 4 °C-treated scWAT and visWAT compared to 30 °C control. Double-headed arrows mark adipocyte diameter. Arrows point to respective positive signals. c–l Quantifications of adipocyte size and positive signals per field of COX4, UCP1, CD31, and alpha SMA of CL-316243- and vehicle-, and 30 °C- and 4 °C- treated scWATs and visWATs (>30 adipocytes per field; n = 10 random fields; n = 5 mice per group). PERI, perilipin; COX4, mitochondrial complex 4; UCP1, uncoupling protein 1. Scale bars, 100 μm. NS, not significant. *P 30 adipocytes per field; n = 10 random fields; n = 5 mice per group). PERI, perilipin; COX4, mitochondrial complex 4; UCP1, uncoupling protein 1. Scale bars, 100 μm. NS, not significant. *P AB-108-C, open histogram) followed by Phycoerythrin-conjugated Anti-Goat IgG Secondary Antibody (Catalog # F0107). View our protocol for Staining Membrane-associated Proteins.

Neutrophil depletion reduces BBB breakdown and increases neovascularization after stroke. f, g Representative confocal images (f) and quantitative analysis of IgG extravascular deposits (g) in the peri-infarct cortex at 14 days in sham-operated mice and mice treated with control antibody or anti-Ly6G antibody (n = 6). One-way ANOVA test was applied with *P < 0.0001 (Sham vs. Isotype), *P = 0.0041 (Isotype vs. Anti-Ly6G). Bar = 15 μm. Image collected and cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/32427863), licensed under a CC-BY license. Not internally tested by R&D Systems.

Increased vascular remodeling by delayed inhibition of NET formation.a–d Representative confocal images (a, c) and quantitative analysis of IgG extravascular deposits (b, d) in the peri-infarct cortex at 14 days. Mice were subjected to stroke and treated with either anti-Ly6G antibody, control antibody, DNase 1, or vehicle starting at 7 days (n = 6), unpaired two-tailed Student’s t-test was applied with *P = 0.0392 (b), *P = 0.0384 (d). Bar = 10 μm. e–l Representative confocal images (e, g) of CD31-positive microvessels and in-vivo multiphoton microscopy images of perfused cortical capillaries with intravenously injected FITC-dextran (i, k) in the peri-infarct cortex at 14 days in mice treated with either anti-Ly6G antibody, control antibody, DNase 1, or vehicle. Bar = 40 μm (e, g) and 100 µm (i, k). Quantification of microvascular density (f, h) and perfused capillary length (j, l) for each group (n = 6), unpaired two-tailed Student’s t-test was applied with *P = 0.00378 (f), *P = 0.0364 (h), *P = 0.0026 (j), *P = 0.0006 (l). Data are presented as mean ± SD. Source data underlying graph b, d, f, h, j, and l are provided as a Source Data file. Image collected and cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/32427863), licensed under a CC-BY license. Not internally tested by R&D Systems.

Neutrophil depletion reduces BBB breakdown and increases neovascularization after stroke. h, j Representative confocal images (h) of CD31-positive microvessels and in-vivo multiphoton microscopy images of perfused cortical capillaries with intravenously injected FITC-dextran (MW = 2000,000 Da) (j) in the peri-infarct cortex at 14 days in mice treated with control antibody or anti-Ly6G antibody, compared with sham-operated mice. Bar = 50 μm (e) and 100 µm (g) Image collected and cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/32427863), licensed under a CC-BY license. Not internally tested by R&D Systems.

Neutrophils accumulate in the brain during all stages of ischemic stroke. f Representative confocal images of Ly6G-labeled neutrophils (green) and CD31-positive microvessels (white) in the peri-infarct cortex of mice at 3 days. Nuclei were visualized with Hoechst. Neutrophils were observed within brain vessels and migrated into the parenchyma. Bar = 40 μm. Independent experiments are repeated at least three times. Image collected and cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/32427863), licensed under a CC-BY license. Not internally tested by R&D Systems.

Reactivity:

Hu, Mu, Rt

Species Glossary

Reactivity: Hu, Mu, Rt

Applications:

, , , ,

Applications: , , , ,

Host:

Goat Polyclonal

Host: Goat Polyclonal

Sizes:

Sample Size Available

Sizes: Sample Size Available

Conjugate:

Unconjugated

Available Conjugates:

Conjugate	Catalog #	Availability	Size	Price
Alexa Fluor 488	FAB3628G-100
Alexa Fluor 488	FAB3628G-025
Allophycocyanin	FAB3628A-100
Allophycocyanin	FAB3628A-025
Biotin	BAF3628
Phycoerythrin	FAB3628P-100
Phycoerythrin	FAB3628P-025

Available Formulations

Formulation	Catalog #	Availability	Price
Carrier Free	AF3628-SP
Carrier Free	FAB3628P-100
Carrier Free	FAB3628P-025
Carrier Free	FAB3628G-100
Carrier Free	FAB3628G-025
Carrier Free	FAB3628A-100
Carrier Free	FAB3628A-025

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AF1924

SOX17 Antibody [Unconjugated]

(3 Reviews)

(458 Publications)

100 μg

AF1924

Western blot shows lysates of BG01V human embryonic stem cells untreated (-) or endoderm differentiated (+). PVDF membrane was probed with 1 µg/mL of Goat Anti-Human SOX17 Antigen Affinity-purified Polyclonal Antibody (Catalog # AF1924) followed by HRP-conjugated Anti-Goat IgG Secondary Antibody (HAF109). A specific band was detected for SOX17 at approximately 55 kDa (as indicated). This experiment was conducted under reducing conditions and using Immunoblot Buffer Group 1.

Endoderm-differentiated D3 mouse embryonic stem cell line was fixed using formaldehyde, resuspended in lysis buffer, and sonicated to shear chromatin. SOX17/DNA complexes were immunoprecipitated using 5 µg Goat Anti-Human SOX17 Antigen Affinity-purified Polyclonal Antibody (Catalog # AF1924) or control antibody (AB-108-C) for 15 minutes in an ultrasonic bath, followed by Biotinylated Anti-Goat IgG Secondary Antibody (BAF109). Immunocomplexes were captured using 50 µL of MagCellect Streptavidin Ferrofluid (MAG999) and DNA was purified using chelating resin solution. Thep21promoter was detected by standard PCR.

SOX17 was detected in immersion fixed B16 mouse melanoma cell line using 10 µg/mL Goat Anti-Human SOX17 Antigen Affinity-purified Polyclonal Antibody (Catalog # AF1924) for 3 hours at room temperature. Cells were stained with the NorthernLights™ 557-conjugated Anti-Goat IgG Secondary Antibody (red; NL001) and counter-stained with DAPI (blue). View our protocol for Fluorescent ICC Staining of Cells on Coverslips.

SOX17 was detected in immersion fixed endoderm differentiated BG01V human embryonic stem cells using 10 µg/mL Goat Anti-Human SOX17 Antigen Affinity-purified Polyclonal Antibody (Catalog # AF1924) for 3 hours at room temperature. Cells were stained with the NorthernLights™ 557-conjugated Anti-Goat IgG Secondary Antibody (red, upper panel; NL001) and counterstained with DAPI (blue, lower panel). View our protocol for Fluorescent ICC Staining of Cells on Coverslips.

Simple Western lane view shows lysates of BG01V human embryonic stem cells untreated (-) or endoderm differentiated (+), loaded at 0.2 mg/mL. A specific band was detected for SOX17 at approximately 59 kDa (as indicated) using 10 µg/mL of Goat Anti-Human SOX17 Antigen Affinity-purified Polyclonal Antibody (Catalog # AF1924) followed by 1:50 dilution of HRP-conjugated Anti-Goat IgG Secondary Antibody (Catalog # HAF109). This experiment was conducted under reducing conditions and using the 12-230 kDa separation system.

Simple Western lane view shows lysates of iBJ6 human induced pluripotent stem cell line untreated (-) or endoderm differentiated (+), loaded at 0.2 mg/mL. A specific band was detected for SOX17 at approximately 58 kDa (as indicated) using 10 µg/mL of Goat Anti-Human SOX17 Antigen Affinity-purified Polyclonal Antibody (Catalog # AF1924) followed by 1:50 dilution of HRP-conjugated Anti-Goat IgG Secondary Antibody (Catalog # HAF109). This experiment was conducted under reducing conditions and using the 12-230 kDa separation system.

Western blot shows lysates of SK‑OV‑3 human ovarian adenocarcinoma cell line, OVCAR‑3 human ovarian carcinoma cell line. PVDF membrane was probed with 2 µg/mL of Goat Anti-Human SOX17 Antigen Affinity-purified Polyclonal Antibody (Catalog # AF1924) followed by HRP-conjugated Anti-Goat IgG Secondary Antibody (Catalog # HAF017). A specific band was detected for SOX17 at approximately 60 kDa (as indicated). This experiment was conducted under reducing conditions and using Western Blot Buffer Group 1.

Generation and characterization of systemic lupus erythematosus (SLE)‐specific human‐induced pluripotent stem cells (hiPSCs). Immunostaining of Otx2 (ectoderm marker), Sox17 (endoderm marker), Brachyury (mesoderm marker) and co‐staining with DAPI (blue) in embryoid bodies (EBs) derived from hiPSCs‐SLE. Scale bar, 50 μm. Image collected and cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/31536674), licensed under a CC-BY licence.

Concordant expression of developmental programs across organoids from human iPSC lines. IF staining of D29 kidney organoid (AS line, ML protocol) for SOX17 and CD31, markers of endothelial cells. Image collected and cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/31784515), licensed under a CC-BY licence.

Activin-A levels during Day 0–1 modulate cardiomyocyte (CM) vs. definitive endoderm (DE) differentiation. Pluripotent H1 ESCs were induced by changing medium to RPMI/B27 (no insulin), including CHIR (7.5 μmol/L) during Day 0–1 and IWP (5 μmol/L) during Days 3–5. Activin-A was included at the indicated levels during Day 0–1. Insulin (4,000 ng/ml) was included after Day 7. a-e shows cells double-immunostained on Day 5 for Oct4 (red) and Sox17 (green); e is a positive control wherein cells were induced to DE with Activin-A (50 ng/ml) and Bmp4 (10 ng/ml) during Days 0–5. f-i shows cells immunostained with MF20 mAb on Day 14 to detect cardiomyocytes. Scale bar: 200 μm. Image collected and cropped by CiteAb from the following publication (//dx.plos.org/10.1371/journal.pone.0118670), licensed under a CC-BY licence.

In vitro and in-TiPSCs. Immunofluorescence staining for Sox17 (endodermal marker), alpha SMA (mesodermal marker), and Nestin (ectodermal marker) in each human TiPSCs1-derived differentiated cell in vitro. Image collected and cropped by CiteAb from the following publication (//dx.plos.org/10.1371/journal.pone.0097397), licensed under a CC-BY licence.

Differentiation of hESCs into definitive endoderm cells by Activin A with CHIR99021. Immunofluorescence analysis of the expression of SOX17 and FoxA2 for Activin A with 2 µM CHIR99021-induced differentiated cells on day 3. Scale bar, 100 µm. Image collected and cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/31601782), licensed under a CC-BY licence.

HIF-1 alpha activates transcription of Sox17. Western blot analysis in control human lung microvascular endothelial cells (HLMVECs) and HLMVECs for which CRISPR/Cas9 was used to delete HIFs. ECs were treated with the HIF prolyl hydroxylase inhibitor DMOG to induce HIF expression. DMOG (1 mM) increased HIF-1 alpha and HIF-2 alpha protein expression in control ECs but not in ECs lacking HIF-1 alpha or HIF-2 alpha . Induction of HIF expression was coupled to Sox17 upregulation. n = 3. Image collected and cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/31073164), licensed under a CC-BY licence.

Features of ESC-INTPSCs intermediate between ESCs and EpiSCs. Expression of OCT3/4 and SOX17. Scale bar, 100 µm. Image collected and cropped by CiteAb from the following publication (//dx.plos.org/10.1371/journal.pone.0095329), licensed under a CC-BY licence.

p38-Mapk14/11 inhibition during murine blastocyst maturation blocks primitive endoderm (PrE) differentiation/maturation. Representative single confocal z-plane micrographs of vehicle control-treated (+DMSO) or p38-Mapk14/11 inhibited (+SB220025) late-blastocyst stage/equivalent embryos, immunofluorescently stained for indicated ICM cell lineage markers (Nanog in green and Gata4, Sox17 and Gata6 in red, plus DAPI DNA stain in blue). Examples of cells classified as TE, PrE and EPI are marked with an asterisk, arrowhead and arrow, respectively. Scale bar, 15 µm. Image collected and cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/27605380), licensed under a CC-BY licence.

Endothelial cell (EC) specific deletion of Sox17 (Sox17EC−/−) in mice prevents endothelial regeneration. Western blot analysis of Sox17 protein expression in isolated ECs obtained from flushed lungs of Sox17EC−/− and control mice. n = 4. Image collected and cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/31073164), licensed under a CC-BY licence.

Characterization of hybrid cells.(A) Both ES-pNSC and pES-NSC hybrid cells are positive for alkaline phosphatase staining (100 ×). (B) RT-PCR analysis of Oct4, Nanog, Sox2, and Nestin expression in fusion partner and reprogrammed hybrid cells. Pluripotency markers, Oct4 and Nanog, which were not expressed in NSCs and pNSCs were expressed in GFP+ fusion hybrid cells. On the other hand, Nestin, which was expressed in NSCs and pNSCs was silenced after forming GFP+ fusion hybrid cells. (C) Immunocytochemistry analysis of Oct4 and Nanog in ES-pNSC and pES-NSC hybrid cells (100 ×). (D)In vitro differentiation of ES-pNSC and pES-NSC hybrid cells into ectoderm (Tuj1), mesoderm (SMA), and endoderm (Sox17) lineages (200 ×). (E) In vivo differentiation potential of ES-pNSC and pES-NSC hybrid cells through teratoma assay. These hybrid cells were contributed to secretory epithelium (ectoderm), cartilage (mesoderm) and gut epithelium (endoderm), which were stained with PAS, Asian blue, and hematoxylin eosin, respectively. Each tissue was indicated by arrow head. Image collected and cropped by CiteAb from the following publication (//dx.plos.org/10.1371/journal.pone.0156491), licensed under a CC-BY license. Not internally tested by R&D Systems.

In vitro and in-TiPSCs.(A): Immunofluorescence staining for Sox17 (endodermal marker), alpha SMA (mesodermal marker), and Nestin (ectodermal marker) in each TiPSCs1-derived differentiated cell in vitro. (B): Gross morphology of representative teratomas derived from TiPSCs1 in vivo (hematoxylin and eosin staining). Image collected and cropped by CiteAb from the following publication (//dx.plos.org/10.1371/journal.pone.0097397), licensed under a CC-BY license. Not internally tested by R&D Systems.

Activation of Sox17 at onset of EC regeneration and Sox17-mediated Cyclin E1 expression. a qPCR analysis of gene expression in sorted CD31+ cells from mTmG-Scl mice before and after injury (12 mg/kg i.p.). Sox17, Vegfr2, and Ccne1 increased significantly at day 2 post-LPS compared to baseline. n = 3. Color scale: the fold change increases from red to white to green color. b Western blot analysis in fresh isolated ECs from wild-type mice and quantification c showed a 5-fold increase in Sox17 protein expression within 1 day following injury compared to baseline and followed by recovery within 3 days post-LPS. n = 3. d, e Western blot analysis of cultured HLMVECs in which Sox17 was overexpressed showed 2.5x fold increase in Cyclin E1 protein expression relative to control cells. n = 3. OE, overexpression. f Representation of the CCNE1 promoter region with Sox17 binding sites (circled numbers) and their sequences. g HLMVECs were retrovirally transduced with Sox17 or control plasmid for 3 days, and Ch-IP assay followed by qPCR was performed to amplify Sox17 binding sites in the CCNE1 promoter. n = 3. h 293T cells were transfected with a Sox17 overexpression plasmid containing CCNE1 luciferase reporter constructs. Luciferase values were normalized to Renilla luciferase control reporter values. A schematic representation of corresponding deletion constructs is presented in the right panel. n = 3 and duplicates per sample. **P < 0.01 and ***P < 0.001. Data are shown as mean ± SEM. Analysis was performed using one-way ANOVA for (c) and two-way ANOVA with Bonferroni post-tests for (e, g, h) Image collected and cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/31073164), licensed under a CC-BY license. Not internally tested by R&D Systems.

Generation and characterization of systemic lupus erythematosus (SLE)‐specific human‐induced pluripotent stem cells (hiPSCs). (A) Dermal fibroblasts derived from patient with SLE were reprogrammed into iPSCs using Sendai virus vectors and three clones (#1, #2 and #3) were characterized. RT‐PCR confirms the loss of transgenes in hiPSCs‐SLE (lanes 1, 2 and 3), presence (lane 4) in infected fibroblasts (ipF‐SLE) and absence of Sendai viral transgenes in parental fibroblasts (pF‐SLE) (lane 5). Full‐length gels are presented in File S2. (B) The hiPSCs‐SLE colonies expressed alkaline phosphatase. Scale bar, 500 μm. (C) RT‐qPCR analysis of pluripotency genes OCT4, NANOG, SOX2 and REX1 was performed in fibroblasts and in hiPSCs derived from patient with SLE. All expression values are normalized to GAPDH and relative donor fibroblasts. Data are mean ± SEM and all statistical analysis was made between hiPSCs‐SLE clones and relative fibroblasts by Student's t test showing P‐values ≤ .05 in each comparison. (D) PluriTest assays combines novelty score (blue) on x‐axis and pluripotency score (red) on y‐axis. hiPSCs‐SLE localize in the red cloud suggesting the empirical distribution of pluripotent cells compared to non‐pluripotent blue cloud. (E) Representative images of M‐FISH staining show normal karyotypes of hiPSCs‐SLE clones. (F) Immunofluorescence analysis of pluripotent stem cell markers Nanog (green), Oct4 (red) and co‐staining with DAPI (blue) in hiPSCs‐SLE. Scale bar, 50 μm. (G) Representative images of floating and adherent EBs derived from hiPSCs‐SLE at differentiation day 8 and 18, respectively. Scale bar, 500 μm. (H) RT‐qPCR results confirm the capability of hiPSCs‐SLE to differentiate into all three germ layers. The expression levels of GATA4, HAND1 and PAX6 in EBs are relative to undifferentiated hiPSCs. All expression values are normalized to GAPDH and relative hiPSCs. Data are mean ± SEM and all statistical analysis was made between EBs‐SLE and relative hiPSCs‐SLE clones by Student's t test showing P‐values ≤ .05 in each comparison. (I) Immunostaining of Otx2 (ectoderm marker), Sox17 (endoderm marker), Brachyury (mesoderm marker) and co‐staining with DAPI (blue) in EBs derived from hiPSCs‐SLE. Scale bar, 50 μm Image collected and cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/31536674), licensed under a CC-BY license. Not internally tested by R&D Systems.

soHIF-1 alpha signaling induces Sox17 expression. a MPO activity of flushed lung sample from mice challenged with LPS (12 mg/kg i.p.) for 6 and 24 h. n = 3. b Western blot analysis of wild-type mice lung before and after LPS-induced injury (12 mg/kg i.p.) and its quantification c showed that HIF-1 alpha protein expression increased within 6 h post-LPS and remained increased until day 2. n = 3. d Western blot analysis in freshly isolated ECs from Hif1afl/fl mice and quantification e showed significantly increased Sox17 protein expression after injury when compared to baseline levels. n = 3. f Western blot analysis in freshly isolated ECs from Hif1aEC−/− mice and quantification g showed no significant difference in Sox17 protein expression before and after injury. n = 3. **P < 0.01 and ***P < 0.001. Data are shown as mean ± SEM. Analysis was performed using one-way ANOVA for (b, c, e, g). Image collected and cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/31073164), licensed under a CC-BY license. Not internally tested by R&D Systems.

PGCLCs derived from hc-iPSCs.Immunofluorescent staining of (A) BLIMP1 (green color) and (B) OCT4 (green color) in 4-day-old EBs cultured with PGC induction medium on hc-iPSC-1, -2, -5 and -6 (third to fifth column), PBMC derived iPSC0102 and iPSC0207 (right two column). H9 hESCs (left column) adapted in 4i medium was also detected for BLIMP1 and OCT4 antibodies. Noted that there was no BLIMP1 signals detected in undifferentiated H9 in 4i condition. All samples were doubly stained with SOX17 antibody (red color) sequentially. DAPI was used for counterstain (blue). Scale bar = 50μm. (C) Quantification of immunofluoresent staining of PGCLCs. BLIMP1/SOX17 and OCT4/SOX17 double positive cells were counted for PGCLCs and normalized with the number of DAPI. Percentage of PGCLCs per EB of all lines was presented and significance was compared with control hESC H9. ** indicates P<0.005. *** indicates P<0.001. Image collected and cropped by CiteAb from the following publication (//dx.plos.org/10.1371/journal.pone.0165715), licensed under a CC-BY license. Not internally tested by R&D Systems.

Activin-A levels during Day 0–1 modulate CM vs. DE differentiation.Pluripotent H1 ESCs were induced by changing medium to RPMI/B27 (no insulin), including CHIR (7.5 μmol/L) during Day 0–1 and IWP (5 μmol/L) during Days 3–5. Activin-A was included at the indicated levels during Day 0–1. Insulin (4,000 ng/ml) was included after Day 7. Panel A, a-e shows cells double-immunostained on Day 5 for Oct4 (red) and Sox17 (green); e is a positive control wherein cells were induced to DE with Activin-A (50 ng/ml) and Bmp4 (10 ng/ml) during Days 0–5. Panel A f-i shows cells immunostained with MF20 mAb on Day 14 to detect cardiomyocytes. Panel B depicts the effect of Activin-A levels during Day 0–1 on cardiomyocyte differentiation at Day 14, determined by flow cytometry using anti-cTnT. Cultures treated with 10 ng/ml Activin-A began to rhythmically contract at Day 6. Cells treated with 50 or 100 ng/ml Activin-A did not beat at any time. Bars indicate the average values combined from multiple experiments. Vertical lines = ±SEM. P-values were calculated by Student’s t-test. The p-value over the bar denoting 10 ng/ml Activin-A is relative to cells treated with CHIR only (0 ng/ml Activin-A), whereas the p-values over the bars denoting 50 and 100 ng/ml Activin-A are relative to cells treated with 10 ng/ml Activin-A. The size bar in Aa, which pertains to panels a-i, = 200 μm. Image collected and cropped by CiteAb from the following publication (//dx.plos.org/10.1371/journal.pone.0118670), licensed under a CC-BY license. Not internally tested by R&D Systems.

Features of ESC-INTPSCs intermediate between ESCs and EpiSCs.(A) Comparison of gene expression in ESCs, EpiSCs, and ESC-INTPSCs. (B) The number of AP-positive colonies formed in the LIF/2i condition four days after 1000 cells were seeded per dish. (C) Proliferation of ESCs, EpiSCs, and ESC-INTPSCs with or without JAKi. (D) Expression of ESRRB and FOXA2. White arrows indicate ESRRB- and FOXA2-double–positive cells. Scale bar, 100 µm. (E) Percentage of ESRRB-positive or ESRRB- and FOXA2-double–positive cells in ESCs, ESC-INTPSCs, and EpiSCs. (F) Expression of OCT3/4 and SOX17. Scale bar, 100 µm. (G) The number of Oct3/4-GFP–positive or –negative colonies derived from single cells at day 4 and 8. Image collected and cropped by CiteAb from the following publication (//dx.plos.org/10.1371/journal.pone.0095329), licensed under a CC-BY license. Not internally tested by R&D Systems.

EC specific deletion of Sox17 (Sox17EC−/−) in mice prevents endothelial regeneration. a Schematic diagram of Sox17fl/fl mice crossed with Scl-CreERT2 to delete Sox17 in ECs. After tamoxifen feeding for five days and rest for another four weeks, mice are challenged with LPS (sub-lethal 8 mg/kg, i.p.) for analysis. b Western blot analysis of Sox17 protein expression in isolated ECs obtained from flushed lungs of Sox17EC−/− and control mice. n = 4. c Quantification of b shows 80% deletion of Sox17 in ECs of Sox17EC−/− mice compared to control mice. n = 4. d Time course of lung transvascular permeability following LPS challenge in Sox17EC−/− and Sox17fl/fl mice. n = 4. While control mouse lungs showed increased endothelial permeability at day 1 post-LPS and then recovered to baseline by day 5, Sox17EC−/− mice showed prolonged endothelial barrier leakiness post-LPS. e Time course of changes in lung transvascular permeability following LPS challenge was also carried out in Sox17fl/fl mice crossed with Cdh5-CreERT2 mice. n = 4. Similar as in d, these Sox17EC−/− mice also showed persistent leakiness post-LPS while control mice fully recovered. f Survival curve of LPS challenge in Sox17EC−/− and control mice. n = 8 per group. At this sub-lethal dose, all control mice survived whereas half of Sox17EC−/− mice died on day 2 post-LPS with increased mortality on day 3. By day 5, the death rate for control mice is 0 while for Sox17EC−/− mice is 60%. g Flow cytometry analysis of CD31+CD45− ECs among whole lung population in mice following injury. n = 4. In contrast to control mice in which CD31+CD45− EC population gradually recovered with day 3 post-LPS after initial loss of ECs, Sox17EC−/− mice showed significantly delayed restoration of ECs post-LPS period. h Quantification of BrdU+ nuclei in each field of 425 μm2 area in flushed lung cryo-sections from mice following injury. n = 4 mice per group and 6 replicates per sample. Slides are co-stained with CD31-AF488, BrdU-APC, and DAPI. At day 3 post-LPS, the control group showed a significantly higher number of BrdU+ECs compared to baseline. However, Sox17EC−/− mice showed markedly reduced level of BrdU+ECs, indicating reduced EC proliferation. i To assess whether expression of Sox17 in ECs can restore lung endothelial integrity, studies were performed in Sox17EC−/− mice to overexpress Sox17 protein. We used a mixture of 50 μg plasmid (mouse Cdh5 promoter—Flag —Sox17) encapsulated in liposomes, which were injected i.v. 3 h after LPS challenge. j At day 3 post-LPS, liposome vector-treated Sox17EC−/− mice showed marked EC barrier leakiness as assessed by lung transvascular permeability of albumin whereas the Sox17-rescued mice showed markedly reduced endothelial permeability. n = 4. OE, overexpression. *P < 0.05, **P < 0.01 and ***P < 0.001. Data are shown as mean ± SEM. Analysis was performed using two-tailed Student’s t-test for (c, j), two-way ANOVA with Bonferroni post-tests for (d, e, g, h) and Log-rank (Mantel-Cox) test for (f) Image collected and cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/31073164), licensed under a CC-BY license. Not internally tested by R&D Systems.

p38-Mapk14/11 inhibition during blastocyst maturation blocks PrE differentiation/maturation. (a) Experimental schema of p38-Mapk14/11 inhibition (+SB220025), plus vehicle control (+DMSO), and the details of antibodies used to analyse ICM cell lineage marker protein expression by immunofluorescence (IF) in late blastocysts (E4.5); Nanog and Gata4 (+DMSO n = 27, +SB220025 n = 33)—green, Nanog and Sox17 (+DMSO n = 18, +SB220025 n = 20)—red, and Nanog and Gata6 (+DMSO n = 24, +SB220025 n = 27)—blue. (b) Representative single confocal z-plane micrographs of vehicle control-treated (+DMSO) or p38-Mapk14/11 inhibited (+SB220025) late-blastocyst stage/equivalent embryos, immunofluorescently stained for indicated ICM cell lineage markers (Nanog in green and Gata4, Sox17 and Gata6 in red, plus DAPI DNA stain in blue). Examples of cells classified as TE, PrE and EPI are marked with an asterisk, arrowhead and arrow, respectively. Scale bar, 15 µm. (c) Pie charts of the relative cell lineage contribution in vehicle control (+DMSO) and p38-Mapk14/11 inhibited (+SB220025) blastocysts as judged by IF to detect the stated ICM lineage marker proteins. Blue, trophectoderm (TE); yellow, epiblast (EPI—ICM exhibiting exclusive Nanog expression); green, primitive endoderm (PrE—ICM exhibiting exclusive Gata4/6 or Sox17 expression, as appropriate); orange, uncommitted ICM cells (exhibiting co-expression of both Nanog and Gata4/6 or Sox17, as appropriate); and grey, ICM cells negative for either assayed marker. (d) Bar charts show average number of cells allocated to each specified ICM lineage, as judged by the indicated IF staining regime employed. Error bars represent s.e.m. and asterisks denote statistical significant differences in cell number between the vehicle control (+DMSO, black bars) and p38-Mapk14/11 inhibited (+SB220025, grey bars) embryo groups, according to two-tailed Student's t-test, with *p < 0.05 and **p < 0.005 confidence intervals. Yellow asterisk denotes increase in cells positively immunofluorescently staining for both EPI and PrE ICM markers using anti-Nanog and anti-Gata6 (an early PrE marker) antibodies. All individual embryo data used in the preparation of this figure are contained within the electronic supplementary material, table S3. Image collected and cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/27605380), licensed under a CC-BY license. Not internally tested by R&D Systems.

Differentiation of hESCs into definitive endoderm cells by Activin A with CHIR99021.a The relative primitive streak (BRACHYRUY, MESP1, and MIXL1) gene expression of the day 1 differentiated cells by adding Activin A (100 ng/mL) with CHIR99021 (2–5 µM) or Wnt3a (25–100 ng/mL) treatments were determined by real-time quantitative PCR (qPCR). None, no WNT signaling pathway activators were used on day 1 for PS differentiation (Activin A only). b, c After 1 day, the medium was changed to Activin A (100 ng/mL) and 1 × CTS-B27 to induce DE differentiation. The definitive endoderm (FOXA2 and SOX17) and mesoderm (MESP2 and HAND1) relative gene expression levels were determined by qPCR. d Immunofluorescence analysis of the expression of SOX17 and FoxA2 for Activin A with 2 µM CHIR99021-induced differentiated cells on day 3. e, f The expression of SOX17 and FoxA2 for Activin A with 2 µM CHIR99021-induced differentiated cells was determined by flow cytometry on day 3. Isotype control antibodies were used as controls. At a specific gene expression, datum points lacking common letters differ, p < 0.05. Data are represented as the mean ± SD. Scale bar, 100 µm Image collected and cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/31601782), licensed under a CC-BY license. Not internally tested by R&D Systems.

HIF-1 alpha activates transcription of Sox17. a Western blot analysis in control HLMVECs and HLMVECs for which CRISPR/Cas9 was used to delete HIFs. ECs were treated with the HIF prolyl hydroxylase inhibitor DMOG to induce HIF expression. DMOG (1 mM) increased HIF-1 alpha and HIF-2 alpha protein expression in control ECs but not in ECs lacking HIF-1 alpha or HIF-2 alpha . Induction of HIF expression was coupled to Sox17 upregulation. n = 3. b Quantification of a showed that protein expression of HIF-1 alpha and HIF-2 alpha in ECs was significantly increased by DMOG treatment. Sox17 showed a 2.5-fold increase in Sox17 expression control DMSO treated ECs. The increase in Sox17 was significantly reduced in HIF-1 alpha -deleted ECs but preserved in HIF-2 alpha -deleted ECs, indicating the importance of HIF-1 alpha in mediating Sox17 expression. n = 3. c Representation of the SOX17 promoter region with 3 HREs indicated by circled numbers, and their respective sequences are displayed. d HLMVECs were exposed to normoxia or 1% O2 (hypoxia) for 8 h. Ch-IP assay followed by qPCR was performed to amplify the HRE regions in the SOX17 promoter. Studies were performed in ECs exposed to either normoxia or hypoxia. n = 3. e 293T cells were transfected with a HIF-1 alpha overexpression plasmid containing SOX17 luciferase reporter constructs. Luciferase values were normalized to Renilla luciferase control reporter values. A schematic representation of corresponding deletion constructs is presented in the right panel. n = 3 and duplicates per sample. Results show that hypoxia activation of SOX17 HRE3 was required for Sox17 expression. **P < 0.01 and ***P < 0.001. Data are shown as mean ± SEM. Analysis was performed using two-way ANOVA with Bonferroni post-tests for (b, d, e) Image collected and cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/31073164), licensed under a CC-BY license. Not internally tested by R&D Systems.

Concordant expression of developmental programs across organoids from four human iPSC lines. a Heatmap of expression patterns for major nephrogenesis markers across organoid differentiation time points (iPSC D0, D7, D15, and D29, averaged across four cell lines, ML protocol) and human adult kidney. Expression values were row-normalized to obtain z-scores; red color indicates positive z-scores. b Canonical (NPHS2) and data-derived (CLDN5) podocyte marker genes superimposed in tSNE plots from D15 organoids (N2 line, ML protocol). c IF staining of D15 kidney organoid (N2 line, ML protocol) for CLDN5 as a marker of early podocyte differentiation derived from the single-cell data. Additional canonical podocyte markers (NPHS1, WT1) and DAPI staining as shown. d IF staining of D29 kidney organoid (AS line, ML protocol) for SOX17 and CD31, markers of endothelial cells. Image collected and cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/31784515), licensed under a CC-BY license. Not internally tested by R&D Systems.

Localisation of Sox17 staining in proliferative and secretory phase endometrial tissue and regulation by hormones. Within the proliferative (A) and secretory (B) endometrium, Sox17 localized to the glandular (open arrowheads) and luminal (closed arrowheads) epithelium, with staining appearing in an irregular, patchy, pattern. Smaller inset boxes show relevant IgG negative controls. Imaged at 20x magnification, scale bar = 50 μm. Treatment of endometrial luminal epithelial (ECC-1) cells with 10−8 M estrogen (estradiol)/10−7 M progesterone (medroxyprogesterone acetate) (, *p < 0.05) resulted in an upregulation of Sox17 protein, when compared to untreated, estradiol only (■, E) treatment groups. (C,D) Data presented as mean ± SEM, n = 5. Representative Western immunoblot shown. Image collected and cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/31664088), licensed under a CC-BY license. Not internally tested by R&D Systems.

Knockdown of SOX17 results in reduced expression in endometrial luminal epithelial cells. Transfection of ECC-1 cells with a CRISPR/Cas9 SOX17 knock down plasmid resulted in decreased SOX17 expression (KD1-5) when compared to untransfected control ECC-1 cells (ECC-1) and ECC-1 cells transfected with a control plasmid (CP). Analysed by Western immunoblot, representative blot shown. Image collected and cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/31664088), licensed under a CC-BY license. Not internally tested by R&D Systems.

Reactivity:

Hu

Species Glossary

Reactivity: Hu

Applications:

, , ,

Applications: , , ,

Host:

Goat Polyclonal

Host: Goat Polyclonal

Sizes:

Sample Size Available

Sizes: Sample Size Available

Conjugate:

Unconjugated

Available Conjugates:

Conjugate	Catalog #	Availability	Size	Price
Allophycocyanin	IC1924A
Biotin	BAF1924
NL557	NL1924R

Available Formulations

Formulation	Catalog #	Availability	Price
Carrier Free	IC1924A
Carrier Free	NL1924R
Carrier Free	AF1924-SP

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NB600-1384

LC3B Antibody - BSA Free

(19 Reviews)

(428 Publications)

0.1 ml

NB600-1384

FTC-133/TPC-1 cells were transfected with HMGB1 shRNA and control shRNA and then pre-treated for 1 h with pepstatin A (PA, 10 uM) and E64D (10 uM) as indicated. Cells were subsequently treated for 3 h with HBSS in continuous presence or absence PA/E64D inhibitors. LC3-I/II levels were assayed by Western blot. Image collected and cropped by CiteAb from the following publication (//jeccr.biomedcentral.com/articles/10.1186/s13046-019-1328-3) licensed under a CC-BY license.

LC3B immunohistochemical staining in CC tissue(A) CC with low LC3B dot like staining. (B) CC with high LC3B dot like staining. (magnification 20X). Image collected and cropped by CiteAb from the following publication (//www.oncotarget.com/lookup/doi/10.18632/oncotarget.17554) licensed under a CC-BY license.

Analysis of LC3B in treated U87-MG (human glioblastoma astrocytoma) lysates using anti- [Catalog # NB600-1384].

Depletion of RARalpha prevents ATRA-induced autophagic flux in SKBR3 cells. LC3B western blotting and quantification of the autophagic activity in control and RARalpha-knockdown SKBR3 cells upon treatment with 1 uM ATRA for 2 days in the presence or absence of BafA for 2 h at 200 nM. LC3B-II expression was normalized to GAPDH and to vehicle-treated control cells. Standard deviations from at least five independent experiments are shown. Image collected and cropped by CiteAb from the following publication (//www.nature.com/articles/cddis2015236), licensed under a CC-BY license.

Human cervical carcinoma (HeLa) and Mouse Neuroblast cells (Neuro2a) were treated with (+) and without (-) 50 uM Chloroquine overnight. Whole cell protein lysates were prepared in 1x Laemmli sample buffer and approximately 10 ug of each lysate (NBP2-49689 and NBP2-49688) was separated on a 4- 20% gel by SDS-PAGE, transferred to 0.2 um PVDF membrane and blocked in 5% nonfat milk in TBST. The membrane was probed with 2 ug/mL anti-LC3 (NB600-1384) and 1 ug/mL anti-alpha tubulin (NB100-690) as a loading control, and detected with the appropriate secondary antibodies using NovaLume Pico Chemiluminescence Substrate (NBP2-61915).

<b>Biological Strategies Validation. </b>Fibroblasts were exposed to 1 mM LLOMe and when indicated pretreated with the autophagy inhibitors 3-methyladenine (5 mM, 1 h) or bafilomycin A1 (25 nM, 30 min). Immunostaining with corresponding colocalization analysis of a) LC3 (green) and galectin-3 (red) and b) LC3 (green) and LAMP2 (red). Merged images show colocalization in yellow, nuclei are stained with DAPI (blue). LLOME = l-leucyl-l-leucine methyl ester, a lysosomal membrane damage compound. Image collected and cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/32409651/) licensed under a CC-BY license.

Confocal analysis of HeLa cells using Rabbit anti-LC3B antibody (Catalog # NB600-1384, 1:5). An Alexa Fluor 488-conjugated Goat to rabbit IgG was used as secondary antibody (green). Actin filaments were labeled with Alexa Fluor 568 phalloidin (red). DAPI was used to stain the cell nuclei (blue).

Immunocytochemical/Immunofluorescent staining of treated U373-MG cells using the HRP conjugate of anti- (Catalog # NB600-1384). The nuclei were stained with DAPI.

Staining of treated U373-MG (human glioblastoma) cells using anti- [Catalog # NB600-1384].

Analysis of U87MG glioma xenografts using anti- [Catalog # NB600-1384]. Image from verified customer review.

LC3B staining in treated U87-MG cultured & subcutaneous tumors.

LC3B staining in glioblastoma multiform tissue.

Lane view shows a specific band for LC3B in 0.5 mg/ml of Neuro2A lysate at a molecular weight of approximately 15 kDa. This experiment was performed under reducing conditions using the 12-230 kDa separation system.

LC3A and LC3B siRNAs specifically block the expression of the LC3A and LC3B proteins, respectively, in A549 cell line (E1,2,3). In E4 the reactivity of and of the LC3B (5F10 antibody) is shown, following silencing of the LC3A or of the LC3B genes, in the A549 cell line. Image collected and cropped by CiteAb from the following publication (//dx.plos.org/10.1371/journal.pone.0137675), licensed under a CC-BY license.

NOD1-dependent induction of autophagy contributes to IRGM and LC3 recruitment to Mtb-containing vesicles. The subcellular localization of autophagy proteins was observed in untreated (A, C) and Tri-DAP treated cells (B, D) using anti-IRGM and anti-LC3 antibodies, which were detected using a secondary antibody coupled to 5 nm gold particles (indicated with arrowheads; TEM X 62,000). Gold particles colocalizing with bacteria were manually counted in 10 macrophages of each condition (E, F) and the differences between the treatments are indicated: ***p < 0.01 and *p < 0.05 vs. Mtb, ++p < 0.01 vs. Tri-DAP, Wilcoxon Rank test. Box plots indicate median and quartiles. Image collected and cropped by CiteAb from the following publication (//bmcpulmmed.biomedcentral.com/articles/10.1186/1471-2466-14-152), licensed under a CC-BY license.

Examples of immunohistochemical stainings; (A) LC3B low, (B) LC3B high, (C) p62 low (any cellular compartment), (D) p62 cytoplasmic low, (E) p62 cytoplasmic/dot like high, nuclear low, (F) p62 cytoplasmic/dot like low, nuclear high, (G) HMGB1 low, (H) HMGB1 high. Objective magnification, 40×.

Simple Western: LC3B Antibody - BSA Free [NB600-1384] - Autolysosome imaging after growth factor deprivation captured the autophagy induction in iPSC-derived astrocytes. (A) Schematic procedures of autophagy induction assay. (B) Representative images of cells and autolysosomes on day 2 with or without growth factor deprivation. (C) Fluorescence counts per well of 96-well plate at 16 h after initiation of assay. Astrocytes were treated with control, starvation and starvation with 10 nM bafilomycin A1 (BafA1) condition. The data were analysed by one-way ANOVA (F [3, 12] = 21.19, p < 0.0001), followed by Tukey's multiple comparisons test. *p < 0.05, **p < 0.01, ****p < 0.001, ns, not significant. Data represent mean +- SD (n = 4, from different experimental replicates). (D) Astrocytes were treated with control, starvation and starvation with 10 nM bafilomycin A1 (BafA1) condition. Cells were lysed and subjected to immunoblot analysis at 16 h after initiation of assay. M.W., molecular-weight size marker. Image collected and cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/38509731 ), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Reactivity:

Hu, Mu, Rt, Po, Ba, Bv, Ca, Pm, Ye, Ze

Species Glossary

Reactivity: Hu, Mu, Rt, Po, Ba, Bv, Ca, Pm, Ye, 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	NB600-1384AF350
Alexa Fluor 405	NB600-1384AF405
Alexa Fluor 488	NB600-1384AF488
Alexa Fluor 532	NB600-1384AF532
Alexa Fluor 594	NB600-1384AF594
Alexa Fluor 647	NB600-1384AF647
Alexa Fluor 700	NB600-1384AF700
Alexa Fluor 750	NB600-1384AF750
Allophycocyanin	NB600-1384APC
Biotin	NB600-1384B
CoraFluor 1	NB600-1384CL1
DyLight 350	NB600-1384UV
DyLight 405	NB600-1384V
DyLight 488	NB600-1384G
DyLight 550	NB600-1384R
DyLight 594	NB600-1384DL594
DyLight 650	NB600-1384C
DyLight 680	NB600-1384FR
DyLight 755	NB600-1384IR
FITC	NB600-1384F
HRP	NB600-1384H
Janelia Fluor 525	NB600-1384JF525
Janelia Fluor 549	NB600-1384JF549
Janelia Fluor 585	NB600-1384JF585
Janelia Fluor 635	NB600-1384JF635
Janelia Fluor 646	NB600-1384JF646
Janelia Fluor 669	NB600-1384JF669
PE	NB600-1384PE
PerCP	NB600-1384PCP
mFluor Violet 450 SE	NB600-1384MFV450
mFluor Violet 500 SE	NB600-1384MFV500
mFluor Violet 610 SE	NB600-1384MFV610

Available Formulations

Formulation	Catalog #	Availability	Price
BSA Free	NB600-1384SS

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NB300-221

GAPDH Antibody (1D4)

(19 Reviews)

(424 Publications)

0.1 ml

NB300-221

Analysis of GAPDH in human nucleus pulposus cell culture (40ug/lane) using anti-GAPDH antibody. LI-COR Odyssey imaging system was used for detection. Western blot image submitted by a verified customer review.

Analysis of of HeLa cells stained with GAPDH antibody, dilution 1:100 (Green). DAPI staining of nuclear DNA (Blue). The GAPDH antibody produces strong cytoplasmic staining of healthy cells.

Analysis of cell line lysates probed with mouse mAb to GAPDH (theoretical molecular weight: 36 kDa), NB300-221, dilution 1:2,000: [1] protein standard, [2] HEK293, [3] HeLa, [4] SH-SY5Y, [5] COS1, [6] NIH-3T3, and [7] C6 cells. The GAPDH antibody reveals a single band at ~37 kDa in all cell lines. GAPDH is a house keeping protein, the level of which is relatively unaffected by most experimental manipulations, and, as a result, this antibody has been widely used as a western blotting loading control.

Human eye cell lysate. Antibody used at 1:500. WB image submitted by a verified customer review.

Analysis of GAPDH in chicken pectoralis major using anti-GAPDH antibody. The primary antibody was used at a dilution of 1:4000, incubated for 1 hour at room temperature. Western blot image submitted by a verified customer review.

Application of the approach to other cell lines. The remainder of the protocol from Figure 2 was then applied using 100 ug/mL digitonin as the optimal cytosolic extract in each case. An aliquot of each extract was then analyzed by 4-12% SDS PAGE followed by Western blotting and probing with antibodies to GAPDH, BiP and Histone H3 (Used as an alternative nuclear marker due to weakness of the Lamin A signal in HeLa cells). Image collected and cropped by CiteAb from the following publication (//bmcresnotes.biomedcentral.com/articles/10.1186/1756-0500-2-243) licensed under a CC-BY license.

Endothelial responses provoked by Hb-lipid interactions are attenuated by sulfide donors. Confluence HAoECs were exposed to the obtained Hb samples for 8 hours and VCAM-1 expression was determined by Western blotting. GAPDH were used as a loading control. Representative experiment, n = 3. Image collected and cropped by CiteAb from the following publication (//www.hindawi.com/journals/omcl/2018/3812568/), licensed under a CC-BY license.

Regulation of cell migration-associated proteins by NCKU-21 in A549 and CL1-5 cells. Protein expressions of matrix metalloproteinase-2 (MMP-2) and MMP-9 were analyzed in A549 cell treated with NCKU-21 for 24 hr. * P < 0.05 and ** P < 0.01, compared to the control group (without NCKU-21 treatment). Image collected and cropped by CiteAb from the following publication (//dx.plos.org/10.1371/journal.pone.0185021), licensed under a CC-BY license.

FerrylHb induces HO-1 and ferritin in EC. Confluent HUVECs grown on 6-well plates were exposed to heme, Hb, metHb, and ferrylHb (100 umol/L heme or as indicated in complete medium containing 15% of FBS). After 4 hours of incubation total RNA was isolated and HO-1 mRNA level was measured by quantitative RT-PCR. For protein expression, HUVECs were solubilized after 8 hours of treatment. HO-1 and ferritin H and L expression was detected by Western blot. Immunoblots were reprobed with GAPDH and are representative of three independent experiments. Results are shown as mean +/- S.D. (n = 3) from one representative experiment of three. Image collected and cropped by CiteAb from the following publication (//www.hindawi.com/journals/omcl/2013/676425/), licensed under a CC-BY license.

SH-SY5Y cells stained with GAPDH antibody NB300-221 (green). Nuclear DNA is stained with Hoechst dye (blue).

Lane view shows a specific band for GAPDH using 0.05 mg/mL Jurkat and MCF-7 cell lysates and antibody at 1:25. Electropherogram image of corresponding Simple Western lanes view at WES molecular weight of 40 kDa. Image reported by internal validation.

Reactivity:

Hu, Mu, Rt, Po, Av, Bv, Ca, Ch, ChHa, Dr, Eq, Fe, Ha, Ma-Op, Pm, Rb, Sh, Ze

Species Glossary

Reactivity: Hu, Mu, Rt, Po, Av, Bv, Ca, Ch, ChHa, Dr, Eq, Fe, Ha, Ma-Op, Pm, Rb, Sh, Ze

Applications:

, , , , , , ,

Applications: , , , , , , ,

Host:

Mouse Monoclonal

Host: Mouse Monoclonal

Sizes:

Sample Size Available

Sizes: Sample Size Available

Conjugate:

Unconjugated

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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.

Reactivity:

Hu, Mu, Rt, Ca, Eq, Fe, Po

Species Glossary

Reactivity: Hu, Mu, Rt, Ca, Eq, Fe, Po

Applications:

, , , , , ,

Applications: , , , , , ,

Host:

Goat Polyclonal

Host: Goat Polyclonal

Sizes:

Sample Size Available

Sizes: Sample Size Available

Conjugate:

Unconjugated

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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 Life Stage

Lysate Protein State

Lysate Subcellular Fraction

Lysate Tissue

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Protein Or Peptide

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