Product Search for hypoxic post translational modification: Novus Biologicals

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NB100-132

HIF-2 alpha/EPAS1 Antibody (ep190b)

(9 Reviews)

(195 Publications)

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NB100-132

Simple Western: HIF-2 alpha/EPAS1 Antibody (ep190b) [NB100-132] - Lane view 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>Biological Strategies Validation. </b>Western Blot: HIF-2 alpha/EPAS1 Antibody (ep190b) [NB100-132] - Analysis of HIF-2 alpha stabilization over time in 791T cells following exposure to hypoxia. Image using the HRP form of this antibody (NB100-132H). Image collected and cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/23785417/) licensed under a CC-BY license.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

Hypoxia inducible factor (HIF)‐2 alpha is expressed in trunk neural crest cells. A,C, Immunostaining of HIF‐2 alpha in sections from trunk axial level of wild‐type chick embryos at HH11, A, HH13, B, and HH18, C. Arrow denotes ventrally migrating HIF‐2 alpha positive cells. D, Co‐immunostaining of HIF‐2 alpha and HNK1 (marker of migrating neural crest) in sections from trunk axial level of wild‐type HH18 chick embryos. Arrows denote migrating cells double positive for the two proteins. E, Sections of HH13 wild‐type embryo immunostained with DAPI for visualization of nuclei and secondary antibody only (donkey anti‐rabbit Alexa Fluor‐546). F, Western blot analysis for detection of HIF‐2 alpha protein at 21% and 1% O2 following siRNA mediated knockdown of HIF‐1 alpha or HIF‐2 alpha . DIP treated cells were used as a positive control and SDHA as loading control. Lanes between 21% and 1% siCTRL were removed from this figure, indicated by the black line. G, Immunohistochemical staining for HIF‐2 alpha in sections of SK‐N‐BE(2)c neuroblastoma cells cultured at 21% or 1% O2. H, Schematic of where oxygen measurements were performed. I, Oxygen saturation in the trunk of chick embryos during development measured ex ovo using microsensor technique. Error bars represent SEM, n ≥ 3 biologically independent replicates for each time point Image collected and cropped by CiteAb from the following open publication (//pubmed.ncbi.nlm.nih.gov/32940375), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Hypoxia inducible factor (HIF)‐2 alpha is expressed in trunk neural crest cells. A,C, Immunostaining of HIF‐2 alpha in sections from trunk axial level of wild‐type chick embryos at HH11, A, HH13, B, and HH18, C. Arrow denotes ventrally migrating HIF‐2 alpha positive cells. D, Co‐immunostaining of HIF‐2 alpha and HNK1 (marker of migrating neural crest) in sections from trunk axial level of wild‐type HH18 chick embryos. Arrows denote migrating cells double positive for the two proteins. E, Sections of HH13 wild‐type embryo immunostained with DAPI for visualization of nuclei and secondary antibody only (donkey anti‐rabbit Alexa Fluor‐546). F, Western blot analysis for detection of HIF‐2 alpha protein at 21% and 1% O2 following siRNA mediated knockdown of HIF‐1 alpha or HIF‐2 alpha . DIP treated cells were used as a positive control and SDHA as loading control. Lanes between 21% and 1% siCTRL were removed from this figure, indicated by the black line. G, Immunohistochemical staining for HIF‐2 alpha in sections of SK‐N‐BE(2)c neuroblastoma cells cultured at 21% or 1% O2. H, Schematic of where oxygen measurements were performed. I, Oxygen saturation in the trunk of chick embryos during development measured ex ovo using microsensor technique. Error bars represent SEM, n ≥ 3 biologically independent replicates for each time point Image collected and cropped by CiteAb from the following open publication (//pubmed.ncbi.nlm.nih.gov/32940375), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Hypoxia inducible factor (HIF)‐2 alpha is expressed in trunk neural crest cells. A,C, Immunostaining of HIF‐2 alpha in sections from trunk axial level of wild‐type chick embryos at HH11, A, HH13, B, and HH18, C. Arrow denotes ventrally migrating HIF‐2 alpha positive cells. D, Co‐immunostaining of HIF‐2 alpha and HNK1 (marker of migrating neural crest) in sections from trunk axial level of wild‐type HH18 chick embryos. Arrows denote migrating cells double positive for the two proteins. E, Sections of HH13 wild‐type embryo immunostained with DAPI for visualization of nuclei and secondary antibody only (donkey anti‐rabbit Alexa Fluor‐546). F, Western blot analysis for detection of HIF‐2 alpha protein at 21% and 1% O2 following siRNA mediated knockdown of HIF‐1 alpha or HIF‐2 alpha . DIP treated cells were used as a positive control and SDHA as loading control. Lanes between 21% and 1% siCTRL were removed from this figure, indicated by the black line. G, Immunohistochemical staining for HIF‐2 alpha in sections of SK‐N‐BE(2)c neuroblastoma cells cultured at 21% or 1% O2. H, Schematic of where oxygen measurements were performed. I, Oxygen saturation in the trunk of chick embryos during development measured ex ovo using microsensor technique. Error bars represent SEM, n ≥ 3 biologically independent replicates for each time point Image collected and cropped by CiteAb from the following open publication (//pubmed.ncbi.nlm.nih.gov/32940375), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Hypoxia inducible factor (HIF)‐2 alpha affects proliferation and self‐renewal capacity. A‐D, Embryo sections from trunk axial level. Proliferating EdU+ cells are green and electroporated cells (morpholinos, A; pCI‐CTRL and pCI‐EPAS1, C) are red. DAPI counterstains nuclei. Only construct targeted neural crest cells (above and outside of dotted line) were quantified (n = 82 [5′‐mispair morpholino] and n = 303 [EPAS1 morpholino], B); n = 211 (pCI‐CTRL) and n = 139 (pCI‐EPAS1), D. Statistical significance calculated using one‐way analysis of variance (ANOVA). E, Relative mRNA expression of EPAS1 in crestosphere cells established from cranial or trunk axial level measured by qRT‐PCR. Expression is presented as mean of n = 4 (cranial) or n = 3 (trunk) biological replicates and error bars represent SEM. Statistical significance calculated using two‐sided student's t test. F,G, Primary sphere assay of crestospheres established from embryos previously electroporated in ovo with non‐ (CTRL) or EPAS1 (EPAS1.1, F, EPAS1.2, G, targeting gRNAs. One cell/well, n = 10 wells/group). Number of spheres were manually counted after 1 week. Statistical significance was calculated by one‐way ANOVA. H, Size of spheres formed in, G. Manual measurements using the ImageJ software to convert to factual unit (μm). Statistical significance was determined by one‐way ANOVA. Inserted photographs of representative spheres from each group Image collected and cropped by CiteAb from the following open publication (//pubmed.ncbi.nlm.nih.gov/32940375), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Hypoxia inducible factor (HIF)‐2 alpha is expressed in trunk neural crest cells. A,C, Immunostaining of HIF‐2 alpha in sections from trunk axial level of wild‐type chick embryos at HH11, A, HH13, B, and HH18, C. Arrow denotes ventrally migrating HIF‐2 alpha positive cells. D, Co‐immunostaining of HIF‐2 alpha and HNK1 (marker of migrating neural crest) in sections from trunk axial level of wild‐type HH18 chick embryos. Arrows denote migrating cells double positive for the two proteins. E, Sections of HH13 wild‐type embryo immunostained with DAPI for visualization of nuclei and secondary antibody only (donkey anti‐rabbit Alexa Fluor‐546). F, Western blot analysis for detection of HIF‐2 alpha protein at 21% and 1% O2 following siRNA mediated knockdown of HIF‐1 alpha or HIF‐2 alpha . DIP treated cells were used as a positive control and SDHA as loading control. Lanes between 21% and 1% siCTRL were removed from this figure, indicated by the black line. G, Immunohistochemical staining for HIF‐2 alpha in sections of SK‐N‐BE(2)c neuroblastoma cells cultured at 21% or 1% O2. H, Schematic of where oxygen measurements were performed. I, Oxygen saturation in the trunk of chick embryos during development measured ex ovo using microsensor technique. Error bars represent SEM, n ≥ 3 biologically independent replicates for each time point Image collected and cropped by CiteAb from the following open publication (//pubmed.ncbi.nlm.nih.gov/32940375), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Reactivity:

Hu, Mu, Rt, Bv, Ha

Species Glossary

Reactivity: Hu, Mu, Rt, Bv, Ha

Applications:

, , , , , , , , , , ,

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

Host:

Mouse Monoclonal

Host: Mouse Monoclonal

Sizes:

Sample Size Available

Sizes: Sample Size Available

Conjugate:

Unconjugated

Available Conjugates:

Validated by:

Validated by:

Conjugate	Catalog #	Availability	Size	Price
Alexa Fluor 350	NB100-132AF350
Alexa Fluor 405	NB100-132AF405
Alexa Fluor 488	NB100-132AF488
Alexa Fluor 532	NB100-132AF532
Alexa Fluor 594	NB100-132AF594
Alexa Fluor 647	NB100-132AF647
Alexa Fluor 700	NB100-132AF700
Alexa Fluor 750	NB100-132AF750
Allophycocyanin	NB100-132APC
Biotin	NB100-132B
CoraFluor 1	NB100-132CL1
DyLight 350	NB100-132UV
DyLight 405	NB100-132V
DyLight 488	NB100-132G
DyLight 550	NB100-132R
DyLight 594	NB100-132DL594
DyLight 650	NB100-132C
DyLight 680	NB100-132FR
DyLight 755	NB100-132IR
FITC	NB100-132F
HRP	NB100-132H
Janelia Fluor 525	NB100-132JF525
Janelia Fluor 549	NB100-132JF549
Janelia Fluor 646	NB100-132JF646
Janelia Fluor 669	NB100-132JF669
PE	NB100-132PE
PerCP	NB100-132PCP
mFluor Violet 450 SE	NB100-132MFV450
mFluor Violet 500 SE	NB100-132MFV500
mFluor Violet 610 SE	NB100-132MFV610

Available Formulations

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

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NB100-137

EGLN1/PHD2 Antibody - BSA Free

(12 Reviews)

(96 Publications)

0.1 mg

NB100-137

Western Blot: EGLN1/PHD2 Antibody [NB100-137] - 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>Genetic Strategies Validation. </b>Simple Western: EGLN1/PHD2 Antibody [NB100-137] - Simple Western lane view shows a specific band for PHD2/HIF Prolyl Hydroxylase 2 in 0.5 mg/mL of hypoxic HeLa cell lysate. This experiment was performed under reducing conditions using the 12-230 kDa separation system.

Western Blot: EGLN1/PHD2 Antibody [NB100-137] - Analysis of PHD2 expression in human prostate cancer cell lines. Western blot image submitted by a verified customer review.

Immunocytochemistry/Immunofluorescence: EGLN1/PHD2 Antibody [NB100-137] - Staining of endogenous PHD2 in U2OS cells. ICC/IF image submitted by a verified customer review.

<b>Genetic Strategies Validation. </b>Western Blot: EGLN1/PHD2 Antibody [NB100-137] - Detection of PHD2 in human glioblastoma cell lysates. Image submitted by a verified customer review.

Western Blot: EGLN1/PHD2 Antibody [NB100-137] - Detection of human PHD2 by Western blot. Recombinant epitope-tagged PHD1, PHD2 or PHD3 (10 ng/lane) or whole cell lysate from HeLa cells. EGLN1/PHD2 antibody used at 1 ug/mL. Detection by chemiluminescence.

Immunohistochemistry: EGLN1/PHD2 Antibody [NB100-137] - EGLN1/PHD2 and EGFR expression levels positively correlate in breast cancer. Processed tissue microarrays of breast cancer biopsies from 313 patients were stained with EGLN1/PHD2 and EGFR antibodies. Four representative immunohistochemistries of human breast cancer with low and high expression of EGLN1/PHD2 and EGFR are shown. Magnification 10x. Image collected and cropped by CiteAb from the following publication (//www.oncotarget.com/article/14241/text/) licensed under a CC-BY license.

Western Blot: EGLN1/PHD2 Antibody [NB100-137] - Exogenous PHD2 has no impact on the gene expression of HIF-1alpha and HIF-2alpha. U87MG cells were transfected with empty vector (pcDNA) or PHD2 plasmid. Twenty-four hours after transfection, PHD2 was detected by immunoblotting. beta-Actin was used as loading control. The results are representative for three independent experiments. Image collected and cropped by CiteAb from the following publication (//www.nature.com/articles/cddis2014295), licensed under a CC-BY license.

Flow Cytometry: EGLN1/PHD2 Antibody [NB100-137] - An intracellular stain was performed on Jurkat cells with NB100-137AF647 (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.

Western Blot: EGLN1/PHD2 Antibody [NB100-137] - Analysis of PHD2 in normoxia (lane 1) or hypoxia (lane 2) treated SK-N-BE(2) cells using anti-EGLN1/PHD2 antibody. Western blot image submitted by a verified customer review.

Immunocytochemistry/Immunofluorescence: EGLN1/PHD2 Antibody [NB100-137] - Staining of PHD2 in HeLa cells with DyLight 488 (green). Nuclei and alpha-tubulin were counterstained with DAPI (blue) and DyLight 550 (red).

Immunohistochemistry: EGLN1/PHD2 Antibody [NB100-137] - Staining of lung vascular endothelium with EGLN1/PHD2 antibody. Image at 40X.

Flow Cytometry: EGLN1/PHD2 Antibody [NB100-137] - Detection of PHD2 in Jurkat cells. One million Jurkat cells were fixed, permeabilized, and stained with 3.0 ug/mL anti-EGLN1/PHD2 antibody in a 150 uL reaction. Isotype control (black), anti-MLL1 (red).

PHD2 and EGFR expression levels positively correlate in breast cancerProcessed tissue microarrays of breast cancer biopsies from 313 patients were stained with PHD2 and EGFR antibodies (cf Materials and methods). Four representative immunohistochemistries of human breast cancer with low and high expression of PHD2 and EGFR are shown. Magnification 10x.

Initial characterization of the OS-9 protein.(A) OS-9 expression in various human cell lines. Equal protein amounts of total cell lysates were used for SDS-PAGE & subsequent Western blotting. For each cell line, two independent samples are shown. Endogenous OS-9 was detected with a polyclonal antibody raised against a peptide corresponding to amino acids 600–667 of isoform 1 of OS-9. (B) Protein stability assay of endogenous OS-9. U2OS cells were treated with the translational inhibitor cycloheximide (100 µM). At indicated time points, whole cell lysates were analysed by immunoblotting. (C) Effect of hypoxia on OS-9 expression. For hypoxia, UT-7 cells were exposed to 1% O2 for 24 h prior to Western blot analysis. To determine any influence of HIF-1 alpha on OS-9 expression under normoxia, cells were incubated with the prolyl hydroxylase inhibitor DMOG (0.5 mM) for 24 h. (D) Protein interaction between OS-9 & PHD2 in vitro. For co-immunoprecipitation, U2OS cells were transiently co-transfected with the plasmids pOS-9-V5 & pPHD2-His, lysed in NP40 buffer, & subjected to immunoisolation with anti-V5 antibody recognizing OS-9 by its V5-tag. OS-9 & its associated proteins were separated by SDS-PAGE & analyzed by Western blot (lane 2). As controls, samples of untransfected (lane 1) cells or cells transfected with a single plasmid (lanes 3–4) were loaded. Representative Western blots are shown for each subfigure. Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/21559462), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

$Cellular localization of OS-9 & PHD2.(A) A lectin gel-shift assay was conducted to test for glycosylated proteins. Total cell lysates of U2OS cells were incubated in the presence or absence of the endoglycosidases EndoH & PNGaseF for 6 h at 37°C. Digest products were separated on a reducing 10% SDS-PAGE gel which contained concanavalin A co-polymerized in the top layer of the separating gel to retard mobility of glycosylated proteins [56]. Glycosylated OS-9 is indicated as ‘g’, deglycosylated OS-9 as ‘d’. (B) Detection of OS-9 & PHD2 in the nuclear fraction. HEK293 cells with & without transfection of the plasmid pcDNA3-OS-9 were separated into nuclear fraction (N) & postnuclear supernatant (PS), the latter containing cytoplasm & organelles. Western blot analysis included BiP, GAPDH & lamin A as typical marker proteins for the ER, the cytoplasm & the nucleus, respectively. (C) Detection of OS-9 & PHD2 in the cytoplasm. HEK293 cells were co-transfected with pcDNA3-OS-9 & pPHD2-V5. For hypoxia, cells were exposed to 3% O2 for 4 h. Cells were treated with 50 µg/ml digitonin & centrifuged to obtain a cytoplasmic (C) & an organelle fraction (O) & subjected to immunoblotting. (D) Isolation of cellular endomembranes. HEK293 cells were lysed mechanically by several passages through a 30½G needle. The postnuclear supernatant was processed further by ultra-centrifugation to separate the organelles (O) from the cytosol (C). High salt treatment (1 M KCl) of the organelle fraction produced a wash fraction (W) that contained dissociated peripheral membrane proteins. For immunoblot analysis of subcellular fractionations, cell aliquots were normalized for cell number prior to loading (B–D). Representative Western blots are shown for each subfigure. Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/21559462), licensed under a CC-BY license. Not internally tested by Novus Biologicals.$

Western Blot: EGLN1/PHD2 Antibody [NB100-137] - Representative picture of western blot in histopathologically unchanged tissue (N) & primary cancerous tissue (C) from patients with CRC. Immunodetection of bands was performed with Rp anti- PHD1, - PHD2, - PHD3 & - FIH Ab, followed by incubation with goat anti-rabbit HRP-conjugated Ab. The membrane was stripped & incubated with Rp anti-GAPDH Ab, followed by incubation with goat anti-rabbit HRP-conjugated Ab. Bands were revealed using SuperSignal West Femto Chemiluminescent Substrate, Thermo Fisher Scientific (Rockford, IL) & Biospectrum® Imaging System 500, UVP Ltd. (Upland, CA). Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/24195777), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: EGLN1/PHD2 Antibody [NB100-137] - OS-9 shows no effect on regulation of HIF-1 alpha .Total cell lysates were used for SDS-PAGE & subsequent Western blotting. To generate nearly anoxic conditions, cells were exposed to an oxygen consuming chemical system or to 1% or 3% O2 for 4 h to generate hypoxia. (A) U2OS, HeLa & Hep3B cells were transiently transfected with the plasmid pOS-9-V5 48 h prior to the experiment. Lamin A & actin were used as loading controls. (B) U2OS cells were subjected to ER stress by incubation either with tunicamycin (1 µg/ml) or thapsigargin (0.5 µg/ml) for 20 h. To detect HIF-1 alpha under normoxia, cells were treated with DMOG (1 mM) for 4 h. A sample of DMSO-only treated cells was loaded to exclude unspecific side effects of the solvent. (C) U2OS cells were transduced with lentiviral construct pLKO.1-shRNA-OS-9 (shOS-9) mediating a stable knockdown of OS-9 expression. Control cells (c) were transduced with plasmid pLKO.1-puro. Representative Western blots are shown for each subfigure. Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/21559462), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: EGLN1/PHD2 Antibody [NB100-137] - Oxygen chemotaxis is independent of the PHD/HIF pathway. a–dHIF1A, HIF2A, HIF1A+HIF2A & PHD2 CRISPR/Cas9 KO clones characterisation, respectively, regarding O2-directed migration. Left panels: immunoblot validation of HIF1A, HIF2A, HIF1A+HIF2A & PHD2 KO clones. To blot HIFs factors (a, b, c), cells were first pre-treated for 5 h with CoCl2 300 µM before protein extraction, a condition that promotes HIF factors accumulation (cf. Supplementary Fig. 8f). Middle panels: cell trajectories under confinement. Red dashed lines indicate the border of the cell cluster at 0 h. Right panels: relative distribution of MCF10A KO clones versus wt cells at the edge of the cluster at 48 h. These experiments demonstrate that HIF factors deletion does not prevent aerotaxis. e, f Tracks & redistribution of wt & PHD2 KO clone silenced for PHD3 (siPHD3) or not (siCTR). g Tracks & redistribution of MCF10A cells treated with DMOG (50 µM) or CoCl2 (50 µM) to inhibit PHDs, or with vehicle only (DMSO). These experiments demonstrate that PHDs do not participate in O2-sensing during aerotaxis. Confinement was applied for 48 h (a–g). Scale bars, 500 µm Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/30382089), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: EGLN1/PHD2 Antibody [NB100-137] - PHD3 expression correlates with a mesenchymal-like morphology in pancreatic ductal adenocarcinoma cell lines.NHF-1 (Fibroblast) MiaPaca2, Panc1, CAPAN1 & BxPC3 cells were harvested for RNA & protein following 24 hours exposure to normoxia (21% O2) or hypoxia (1% O2). (A) Phase-contrast images at 10× magnification were taken of MiaPaca2 (mesenchymal-like) morphology & BxPC3 cells (differentiated, epithelial morphology) under normoxic conditions. (B) PHD3 mRNA expression was determined by qRT-PCR & graphed relative to BxPC3 in normoxia. All samples were normalized to 18S rRNA & graphed as expression relative to BxPC3-Vec Normoxia (lane 1). n = 3, error bars = 1 S.D. (C) Whole cell lysate was resolved by SDS-PAGE & blotted for beta -tubulin PHD3 & PHD2. N = normoxia, H = hypoxia (1% O2). Image collected & cropped by CiteAb from the following publication (//dx.plos.org/10.1371/journal.pone.0083021), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: EGLN1/PHD2 Antibody [NB100-137] - Iron supplementation restores HIF-1 alpha levels to normal following ATP6V0E1 inhibition in HeLa cells. (A) Schematic diagram of the multimeric V-ATPase complex. (B) Chemical inhibition of V-ATPase by 10 nM BafA treatment for 24 h, increased HIF-1 alpha levels in HIF alpha -GFPODD reporter cells. Treatment with 100 μM Fe (III) citrate significantly reduced the elevated HIF-1 alpha levels associated with loss of ATP6V0E1 (1 × 106 cells per sample harvested & analyzed; N = 2). (C) Knock-down of ATP6V0E1 subunit with three different CRISPR-Cas9 guides resulted in significant upregulation of HIF-1 alpha levels in HIF alpha -GFPODD reporter cells. Co-treating cells with 100 μM Fe (III) citrate led to a reduction in HIF-1 alpha levels across the three ATP6V0E1 depleted cells. phd2 was knocked down as a control & treatment with 100 μM Fe (III) citrate did not result in reduction of HIF-1 alpha levels (1 × 106 cells per sample harvested & analyzed; N = 2). FACs plot shown is a representative image of two biological repeats performed. (D) Immunoblot analysis for HIF-1 alpha & PHD2 levels in HIF alpha -GFPODD reporter cells with either ATP6V0E1 or PHD2 depleted or treated with 10 nM BafA. The cells were treated with 100 μM Fe (III) citrate for 24 h. beta actin was used as a control. Results validated findings observed by flow cytometry, whereby HIF-1 alpha levels were upregulated following ATP6V0E1 knock-down or inhibition & levels were re-normalized upon Fe (III) citrate treatment. Treatment of Fe (III) citrate in PHD2 depleted cells did not alter HIF-1 alpha levels. All experiments were performed in biological duplicate. Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/32984302), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: EGLN1/PHD2 Antibody [NB100-137] - PHD3 depletion stabilizes hypoxic p27 expression by increasing p27 half-life. a Cell cycle arrest at G0 & subsequent release shows an increase of p27 expression in siPHD3 exposed cells. b Quantification for p27 expression under PHD3 depletion at indicated time points after cell cycle release in HeLa & 786-O cells. Asterisk indicates significant difference (p < 0,05; n = 3). c Cell cycle arrest at G0 & inhibition of protein synthesis with cycloheximide indicate increased p27 stability in PHD3 depleted HeLa cells. d Quantification of p27 expression using siPHD3 or control at indicated time points. Four independent experiments (± SEM) are shown (p < 0,05; n = 4). e Analysis of p27 stability in 786–0 cells by cycloheximide chase during reoxygenation after 24 h hypoxia demonstrates markedly increased half-life of p27 upon PHD3 depletion Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/26223520), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Reactivity:

Hu, Mu, Rt, Pm, Pm-Or, Rb, RM

Species Glossary

Reactivity: Hu, Mu, Rt, Pm, Pm-Or, Rb, RM

Applications:

, , , , , , ,

Applications: , , , , , , ,

Host:

Rabbit Polyclonal

Host: Rabbit Polyclonal

Conjugate:

Unconjugated

Available Conjugates:

Validated by:

Validated by:

Conjugate	Catalog #	Availability	Size	Price
Alexa Fluor 350	NB100-137AF350
Alexa Fluor 405	NB100-137AF405
Alexa Fluor 488	NB100-137AF488
Alexa Fluor 532	NB100-137AF532
Alexa Fluor 594	NB100-137AF594
Alexa Fluor 647	NB100-137AF647
Alexa Fluor 700	NB100-137AF700
Alexa Fluor 750	NB100-137AF750
Alexa Fluor Plus 488	NB100-137AFP488
Alexa Fluor Plus 555	NB100-137AFP555
Alexa Fluor Plus 594	NB100-137AFP594
Alexa Fluor Plus 647	NB100-137AFP647
Alexa Fluor Plus 680	NB100-137AFP680
Allophycocyanin	NB100-137APC
Biotin	NB100-137B
CoraFluor 1	NB100-137CL1
DyLight 350	NB100-137UV
DyLight 405	NB100-137V
DyLight 488	NB100-137G
DyLight 550	NB100-137R
DyLight 594	NB100-137DL594
DyLight 650	NB100-137C
DyLight 680	NB100-137FR
DyLight 755	NB100-137IR
FITC	NB100-137F
HRP	NB100-137H
Janelia Fluor 525	NB100-137JF525
Janelia Fluor 549	NB100-137JF549
Janelia Fluor 646	NB100-137JF646
Janelia Fluor 669	NB100-137JF669
PE	NB100-137PE
PerCP	NB100-137PCP
mFluor Violet 450 SE	NB100-137MFV450
mFluor Violet 500 SE	NB100-137MFV500
mFluor Violet 610 SE	NB100-137MFV610

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