EGLN3/PHD3 Antibody - BSA Free

Knockdown Validated: EGLN3/PHD3 Antibody [NB100-139] - PHD3 is required for SCC cell survival in prolonged hypoxia. PHD3 is induced in hypoxia. Knock-down of PHD3 expression with siRNA (siPHD3) is specific and does not affect PHD2 or HIF-1 alpha expression. siScr = scrambled control siRNA. Image collected and cropped by CiteAb from the following publication (//doi.org/10.1371/journal.pone.0014617), licensed under a CC-BY license.

Immunohistochemistry-Paraffin: EGLN3/PHD3 Antibody [NB100-139] - Analysis of a formalin fixed tissue section of human renal cell carcinoma (clear cell type) using rabbit polyclonal EGLN3/PHD3 antibody with HRP-DAB detection and hematoxylin counterstaining. The antibody generated a strong nuclear staining of PHD3 primarily in the cancer cells while the stromal cells were largely negative for this protein.

Knockdown Validated: EGLN3/PHD3 Antibody [NB100-139] - EGLN3/PHD3 depletion increases S10 phosphorylation of p27 in hypoxia. S10 phosphorylated form of p27 is strongly induced in EGLN3/PHD3 depleted cells. Image collected and cropped by CiteAb from the following publication (//molecular-cancer.biomedcentral.com/articles/10.1186/s12943-015-0410-5) licensed under a CC-BY license.

Immunocytochemistry/Immunofluorescence: EGLN3/PHD3 Antibody [NB100-139] - EGLN3/PHD3 antibody was tested in HeLa cells with Dylight 488 (green). Nuclei and alpha-tubulin were counterstained with DAPI (blue) and Dylight 550 (red).

Knockdown Validated: EGLN3/PHD3 Antibody [NB100-139] - BxPC3 cells stably transduced with retrovirus containing PHD3Wt (BxPC3-Wt), Vector (BxPC3-Vec) or anti-PHD3 shRNA (BxPC3-KD) were harvested for RNA and protein following 24 hours exposure to normoxia (21% O2) or hypoxia (1% O2). Whole cell lysate from BxPC3-Wt, BxPC3-Vec and BxPC3-KD cells following 24 hours exposure to normoxia (N) or hypoxia (H) was resolved by SDS-PAGE and blotted for actin (top) and PHD3 (bottom). PHD3 band intensity was quantified relative to actin in each lane and then normalized to BxPC3-Vec (N). Relative band intensity is indicated below the figure. NS = non-specific band running just above PHD3 band. Data is representative of >3 biological replicates. Image collected and cropped by CiteAb from the following publication (//doi.org/10.1371/journal.pone.0083021), licensed under a CC-BY license.

Western Blot: EGLN3/PHD3 Antibody [NB100-139] - The HIF-1 response is not impaired in prostate and breast carcinoma cell lines that lack PHD3. Melanoma, breast and prostate cancer cell lines with methylated PHD3 promoters (MB-435, PC-3) and non-methylated promoters (MCF7, DU 145) were subjected to hypoxia (1% O2) or normoxia for 24 hours. Thirty micrograms of whole cell lysate was western blotted for the presence of PHD3; actin was used as a loading control. Image collected and cropped by CiteAb from the following publication (//doi.org/10.1371/journal.pone.0014617), licensed under a CC-BY license.

Western Blot: EGLN3/PHD3 Antibody [NB100-139] - Analysis of HeLa lysates using NB100-139. Image courtesy of Dr Gregg Semenza (The Johns Hopkins University School of Medicine, Baltimore, MD USA)

• Reactivity: Hu, Mu, Rt
• Applications: WB, ChIP, ICC/IF, IHC, IP, MS, KD, MS
• Clonality: Polyclonal
• Host: Rabbit
• Conjugate: Unconjugated
• Format: BSA Free
• Concentration: 1 mg/ml

EGLN3/PHD3 Antibody - BSA Free Summary

• Immunogen: Synthetic peptide corresponding to residues between 50-100 of human PHD3/HIF Prolyl Hydroxylase 3 using the numbering given in entry NP_071356.1 (GeneID 112399).
• Isotype: IgG
• Clonality: Polyclonal
• Host: Rabbit
• Gene: EGLN3
• Purity: Immunogen affinity purified

Applications/Dilutions

• Dilutions:
  • Chromatin Immunoprecipitation reported in scientific literature (PMID 24367580)
  • Electron Microscopy reported in scientific literature (PMID 17003483)
  • Immunocytochemistry/Immunofluorescence 1:500
  • Immunohistochemistry 1:250-1:1000
  • Immunohistochemistry-Paraffin 1:250-1:1000
  • Immunoprecipitation
  • Knockdown Validated
  • Mass Spectrometry
  • Western Blot 1:1000-1:2000
• Application Notes: In WB, PHD3 band can be seen at 27-30 kDa molecular weight range. Mass Spectrometry reported in scientific literature (PMID:34426491).
• Theoretical MW: 28 kDa.
  Disclaimer note: The observed molecular weight of the protein may vary from the listed predicted molecular weight due to post translational modifications, post translation cleavages, relative charges, and other experimental factors.
• Control:

  EGLN3/PHD3 Overexpression Lysate

Reactivity Notes

Mouse reactivity reported in scientific literature (PMID: 24037093)

Packaging, Storage & Formulations

• Storage: Store at 4C short term. Aliquot and store at -20C long term. Avoid freeze-thaw cycles.
• Buffer: PBS
• Preservative: 0.05% Sodium Azide
• Concentration: 1 mg/ml
• Purity: Immunogen affinity purified

Alternate Names for EGLN3/PHD3 Antibody - BSA Free

• EC 1.14.11
• EC 1.14.11.-
• EGL nine (C.elegans) homolog 3
• egl nine homolog 3 (C. elegans)
• egl nine homolog 3
• egl nine-like protein 3 isoform
• EGLN3
• FLJ21620
• HIF prolyl hydroxylase 3
• HIFPH3 MGC125998
• HIFPH3
• HIF-PH3
• HIF-prolyl hydroxylase 3
• HPH-1
• HPH-3
• Hypoxia-inducible factor prolyl hydroxylase 3
• MGC125999
• pdh3
• PHD3
• Prolyl hydroxylase domain-containing protein 3

Background

HIF prolyl 4-hydroxylases (PHDs) are prolyl hydroxylase domain-containing enzymes (PHD1/ Egln2, PHD2/ Egln1, PHD3/ Egln3, and P4H-TM) that are known for their role in mediating physiological responses to hypoxic stress via modulation of HIF-1 alpha expression levels. The HIF-1 alpha subunit is regulated by hydroxylation, both by a family of PHDs leading to ubiquitination and proteasomal degradation, and by transcriptional inactivation following asparaginyl hydroxylation by FIH (factor inhibiting HIF). When oxygen levels are normal, HIF Prolyl Hydroxylase 3 (HIF-PH3/ PHD3/ Egln3) hydroxylates a specific proline found in HIF-1 alpha's NODD/CODD domains and also hydroxylates HIF-2 alpha with preference for CODD site for HIF1A/HIF2A. Once hydroxylated, HIFs undergo proteasomal degradation via von Hippel-Lindau (VHL) ubiquitination complex. Upon hypoxic trigger, the hydroxylation reaction is tempered which allows HIFs to escape the degradation process leading to their nuclear translocation, heterodimerization with HIF1B, and increased expression of hypoxy-inducible genes. HIF-PH3 is the most important isozyme in limiting physiological activation of HIFs (particularly HIF2A) in hypoxia. Besides HIFs, in hypoxic conditions HIF-PH3 also hydroxylates PKM (thereby limiting glycolysis) and hydroxylates/regulates the stability of ADRB2. In cardiomyocytes, HIF-PH3 inhibits Bcl2's anti-apoptotic effect by disrupting the BAX-BCL2 complex, and in neurons, HIF-PH3 has a NGF-induced proapoptotic effect mediated via CASP3 activity regulation. HIF-PH3 also plays a crucial role in DNA damage response by hydroxylating TELO2, promoting its interaction with ATR which is required for activation of the ATR/CHK1/p53 pathway.

Limitations

This product is for research use only and is not approved for use in humans or in clinical diagnosis. Primary Antibodies are guaranteed for 1 year from date of receipt.

Publications for EGLN3/PHD3 Antibody (NB100-139)(49)

Publications using NB100-139

• Saggese P, Pandey A, Fung E et al. Glucose deprivation promotes pseudo-hypoxia and de-differentiation in lung adenocarcinoma bioRxiv : the preprint server for biology 2023-02-01 [PMID: 36778362] (WB, Human)
  
  Details:
  Dilution used in WB 1:500
• Seike K, Kiledal A, Fujiwara H et al. Ambient oxygen levels regulate intestinal dysbiosis and GVHD severity after allogeneic stem cell transplantation Immunity 2023-02-01 [PMID: 36736321] (WB, Mouse)
  
  Details:
  Dilution used 1:1000
• Ciminera AK, Shuck SC, Termini J Elevated glucose increases genomic instability by inhibiting nucleotide excision repair Life science alliance 2021-10-01 [PMID: 34426491] (MS, Human)
• Nasteska D, Cuozzo F, Viloria K Et Al. Prolyl-4-hydroxylase 3 maintains beta-cell glucose metabolism during fatty acid excess in mice JCI insight 2021-07-15 [PMID: 34264866] (IHC-P)
• Ju S, Lim L, Wi K et al. LRP5 Regulates HIF-1 alpha Stability via Interaction with PHD2 in Ischemic Myocardium International Journal of Molecular Sciences 2021-06-19 [PMID: 34205318] (IP, Rat)
• Shi M, Dai WQ, Jia RR et al. APCCDC20-mediated degradation of PHD3 stabilizes HIF-1a and promotes tumorigenesis in hepatocellular carcinoma Cancer Lett 2020-10-09 [PMID: 33039559] (WB, IP, Mouse)
• Yoon, H, Spinelli, J B Et al. PHD3 Loss Promotes Exercise Capacity and Fat Oxidation in Skeletal Muscle. Cell Metab 2020-08-04 [PMID: 32663458] (WB, Human)
• Kozlova N, Mennerich D, Samoylenko A et al. The pro-oncogenic adaptor CIN85 inhibits hypoxia-inducible factor prolyl hydroxylase-2. Cancer Res 2019-05-31 [PMID: 31142511] (WB, Human)
• Miikkulainen P, Hogel H, Seyednasrollah F et al. Hypoxia-inducible factor (HIF)-prolyl hydroxylase 3 (PHD3) maintains high HIF2A mRNA levels in clear cell renal cell carcinoma. J. Biol. Chem. 2019-01-07 [PMID: 30617181] (WB, Human)
• Yano H, Sakai M, Matsukawa T et al. PHD3 regulates glucose metabolism by suppressing stress-induced signalling and optimising gluconeogenesis and insulin signalling in hepatocytes. Sci Rep 2018-09-24 [PMID: 30250231] (WB, Mouse, Human)
• Pan H, Strickland A, Madhu V et al. RNA binding protein HuR regulates extracellular matrix gene expression and pH homeostasis independent of controlling HIF-1a signaling in nucleus pulposus cells Matrix Biol. 2018-08-06 [PMID: 30092282] (WB)
• Akat F, Ficicilar H, Durak A et al. Intermittent hypoxia induces beneficial cardiovascular remodeling in left ventricular function of type 1 diabetic rat. Anatol J Cardiol 2018-04-01 [PMID: 29615543] (Rat)
• Bryant JD, Brown MC, Dobrikov MI et al. Regulation of HIF-1A during Hypoxia by DAP5-Induced Translation of PHD2. Mol. Cell. Biol. 2018-03-12 [PMID: 29530922] (WB)
• Miikkulainen P, Hogel H, Rantanen K et al. HIF prolyl hydroxylase PHD3 regulates translational machinery and glucose metabolism in clear cell renal cell carcinoma Cancer Metab 2017-07-06 [PMID: 28680592] (WB, Human)
• Bruno T, Valerio M, Casadei L et al. Che-1 sustains hypoxic response of colorectal cancer cells by affecting Hif-1alpha stabilization. J. Exp. Clin. Cancer Res. 2017-02-18 [PMID: 28214471] (WB, Human)
• Oh ET, Kim JW, Kim JM et al. NQO1 inhibits proteasome-mediated degradation of HIF-1a. Nat Commun. 2016-12-14 [PMID: 27966538] (Mouse)
• Barnes EA, Chen CH, Sedan O, Cornfield DN. Loss of smooth muscle cell hypoxia inducible factor-1a underlies increased vascular contractility in pulmonary hypertension FASEB J. 2017-02-01 [PMID: 27811062] (WB, Human)
• Adam MG, Matt S, Christian S et al. SIAH ubiquitin ligases regulate breast cancer cell migration and invasion independent of the oxygen status. Cell Cycle. 2015-12-02 [PMID: 26654769] (WB, Human)
• Iriondo O, Rabano M, Domenici G et al. Distinct breast cancer stem/progenitor cell populations require either HIF1alpha or loss of PHD3 to expand under hypoxic conditions. Oncotarget 2015-10-13 [PMID: 26372732] (WB)
• Hogel H, Miikkulainen P, Bino L, Jaakkola PM. Hypoxia inducible prolyl hydroxylase PHD3 maintains carcinoma cell growth by decreasing the stability of p27. Mol. Cancer. 2015-07-30 [PMID: 26223520] (ICC/IF, WB, Human)
  
  Details:
  PHD1 antibody used for WB on 786-O cell under hypoxia (1% O2) and normoxia (21 % O2) by siPHD1 (Figure 1C).
• Li J, Yuan W, Jiang S et al. Prolyl-4-hydroxylase Domain Protein 2 Controls NF-kB/p65 Transactivation and Enhances the Catabolic Effects of Inflammatory Cytokines on cells of the Nucleus Pulposus J. Biol. Chem. 2015-01-29 [PMID: 25635047] (WB, Rat, Human)
• Vogler M, Zieseniss A, Hesse AR et al. Pre- and post-conditional inhibition of prolyl-4-hydroxylase domain enzymes protects the heart from an ischemic insult Pflugers Arch. 2015-01-13 [PMID: 25578858] (IHC-P, Mouse)
  
  Details:
  PHD3/HIF Prolyl Hydroxylase 3 antibody used at 1:10,000 dilution for IHC-P application on heart sections from C57BL/6 mice after 1, 6, and 24 h of treatment (40-fold magnification) with ICA, a specific PHD inhibitor 2-(1-chloro-4-hydroxyisoquinoline-3-carboxamido) acetate - two doses of 40 mg/kg BW ICA or vehicle i.p., either 6 and 1 h before myocardial infarction or 1 and 5 h after myocardial infarction (Fig 3).
• Place TL, Nauseef JT, Peterson MK et al. Prolyl-4-Hydroxylase 3 (PHD3) Expression Is Downregulated during Epithelial-to-Mesenchymal Transition. PLoS One 2013-12-18 [PMID: 24367580] (IP, WB, Human)
• Rawluszko AA, Bujnicka KE, Horbacka K et al. Expression and DNA methylation levels of prolyl hydroxylases PHD1, PHD2, PHD3 and asparaginyl hydroxylase FIH in colorectal cancer. BMC Cancer. 2013-11-06 [PMID: 24195777] (WB, Human)
• Taniguchi CM, Finger EC, Krieg AJ et al. Cross-talk between hypoxia and insulin signaling through Phd3 regulates hepatic glucose and lipid metabolism and ameliorates diabetes. Nat Med. 2013-10-01 [PMID: 24037093] (WB, Mouse)
• Galan-Cobo A, Sanchez-Silva R, Serna A et al. Cellular overexpression of aquaporins slows down the natural HIF-2a degradation during prolonged hypoxia. Gene. 2013-07-13 [PMID: 23545307] (WB, Rat)
• Fujita N, Markova D, Anderson DG, Chiba K, Toyama Y, Shapiro IM, Risbud MV. Expression of Prolyl Hydroxylases (PHDs) Is Selectively Controlled by HIF-1 and HIF-2 Proteins in Nucleus Pulposus Cells of the Intervertebral Disc: DISTINCT ROLES OF PHD2 AND PHD3 PROTEINS IN CONTROLLING HIF-1? ACTIVITY IN HYPOXIA. J Biol Chem;287(20):16975-86. 2012-05-11 [PMID: 22451659] (WB, ICC/IF, Rat, Human)
• Hogel H, Rantanen K, Jokilehto T et al. Prolyl hydroxylase PHD3 enhances the hypoxic survival and G1 to S transition of carcinoma cells. PLoS One. 2011-01-01 [PMID: 22087251] (WB, Human)
• Anderson K, Nordquist KA, Gao X et al. Regulation of Cellular Levels of Sprouty2 by Prolyl Hydroxylase Domain Proteins and von-Hippel Lindau Protein. The Journal of Biological Chemistry. 2011-10-17 [PMID: 22006925]
• Luo W, Hu H, Chang R et al. Pyruvate Kinase M2 is a PHD3-Stimulated Coactivator for Hypoxia-Inducible Factor 1. Cell;145(5):732-44. 2011-05-27 [PMID: 21620138]
• Di Fonzo MM, Pelletier F, Clutton-Brock TH et al. The population growth consequences of variation in individual heterozygosity. PLoS One 2011-01-01 [PMID: 21611172]
• Place TL, Fitzgerald MP, Venkataraman S et al. Aberrant promoter CpG methylation is a mechanism for impaired PHD3 expression in a diverse set of malignant cells. PLoS One. 2011-01-01 [PMID: 21297970]
• Luukkaa M, Jokilehto T, Kronqvist P et al. Expression of the cellular oxygen sensor PHD2 (EGLN-1) predicts radiation sensitivity in squamous cell cancer of the head and neck. Int J Radiat Biol. 2009-10-01 [PMID: 19639506]
• Xie L, Xiao K, Whalen EJ et al. Oxygen-Regulated {beta}2-Adrenergic Receptor Hydroxylation by EGLN3 Ubiquitylation by pVHL. Sci Signal;2(78):ra33-. 2009-01-01 [PMID: 19584355]
• Chan DA, Kawahara TL, Sutphin PD et al. Tumor vasculature is regulated by PHD2-mediated angiogenesis and bone marrow-derived cell recruitment. Cancer Cell 2009-06-02 [PMID: 19477431]
• Schodel J, Klanke B, Weidemann A et al. HIF-prolyl hydroxylases in the rat kidney: physiologic expression patterns and regulation in acute kidney injury. Am J Pathol 2009-05-01 [PMID: 19349364]
• Wong W, Goehring AS, Kapiloff MS et al. mAKAP Compartmentalizes Oxygen-Dependent Control of HIF-1{alpha}. Sci Signal;1(51):ra18-. 2008-01-01 [PMID: 19109240]
• Newpher TM, Ehlers MD. Glutamate receptor dynamics in dendritic microdomains. Neuron 2008-05-22 [PMID: 18498731] (Human)
• Ginouves A, Ilc K, Macias N, Pouyssegur J, Berra E. PHDs overactivation during chronic hypoxia desensitizes HIFalpha and protects cells from necrosis. Proc Natl Acad Sci U S A;105(12):4745-50. 2008-03-25 [PMID: 18347341]
• Rantanen K, Pursiheimo J, Hogel H, Himanen V, Metzen E, Jaakkola PM. Prolyl Hydroxylase PHD3 Activates Oxygen-dependent Protein Aggregation. Mol Biol Cell;19(5):2231-40. 2008-05-01 [PMID: 18337469]
• Lomb DJ, Straub JA, Freeman RS. Prolyl hydroxylase inhibitors delay neuronal cell death caused by trophic factor deprivation. J Neurochem 2007-12-01 [PMID: 17760870] (Rat)
• Fu, J, Menzies, K, Freeman, R S, Taubman, M B. EGLN3 prolyl hydroxylase regulates skeletal muscle differentiation myogenin protein stability, M608748200. J Biol Chem282(17):12410-8. 2007-04-27 [PMID: 17344222]
• Khan Z, Michalopoulos GK, Stolz DB. Peroxisomal localization of hypoxia-inducible factors and hypoxia-inducible factor regulatory hydroxylases in primary rat hepatocytes exposed to hypoxia-reoxygenation. Am J Pathol 2006-10-01 [PMID: 17003483] (EM, ICC/IF, WB, IHC-P, Rat)
• Li N et al. Expression actions of HIF prolyl-4-hydroxylase in the rat kidneys. Am J Physiol Renal Physiol 292: F207 - F216. 2007-01-01 [PMID: 16885149]
• Asikainen, T M, Waleh, N S, Schneider, B K, Clyman, R I, White, C W. Enhancement of angiogenic effectors through hypoxia-inducible factor in preterm primate lung in vivo, 291, L588-595. Am J Physiol Lung Cell Mol Physiol. 291(4):L588-95. 2006-10-01 [PMID: 16679381]
• Jokilehto T, Rantanen K, Luukkaa M et al. Overexpression and nuclear translocation of hypoxia-inducible factor prolyl hydroxylase PHD2 in head and neck squamous cell carcinoma is associated with tumor aggressiveness. Clin Cancer Res 2006-02-01 [PMID: 16489060]
• Cervera AM, Apostolova N, Luna-Crespo F et al. An alternatively spliced transcript of the PHD3 gene retains prolyl hydroxylase activity. Cancer Lett 2006-02-20 [PMID: 16473674]
• Asikainen, TM et al. Activation of hypoxia-inducible factors in hyperoxia through prolyl 4-hydroxylase blockade in cells explants of primate lung. PNAS 102(29): 10212-10217. 2005-01-01 [PMID: 16009933] (WB, Human)
• Dalgard CL, Lu H, Mohyeldin A, Verma A. Endogenous 2-oxoacids differentially regulate expression of oxygen sensors. Biochem J 2004-06-01 [PMID: 14984367] (Human)

Reviews for EGLN3/PHD3 Antibody (NB100-139) (2)

Average Rating: 5

(Based on 2 reviews)

We have 2 reviews tested in 2 species: Human, Mouse.

reviewed by: Kavitha Subramanian Vignesh WB Mouse 05/08/2016

Summary

• Application: Western Blot
• Species: Mouse

reviewed by: Weibo Luo WB Human 06/24/2010

Summary

• Application: Western Blot
• Sample Tested: HeLa cell lysates, Sample Amount: 20 ug
• Species: Human

Bioinformatics

• Gene Symbol: EGLN3
• Entrez:
  • 112399(Human)
  • 112407(Mouse)
  • 54702(Rat)
• Uniprot:
  • Q9H6Z9()