HIF Prolyl Hydroxylases Antibody Pack

Simple Western: HIF Prolyl Hydroxylases Antibody Pack [NB100-903PHD] - Simple Western lane view shows a specific band for PHD2/HIF Prolyl Hydroxylase 2 in 0.5 mg/mL of Hypoxic HeLa lysate. This experiment was performed under reducing conditions using the 12-230 kDa separation system. EGLN1/PHD2 Antibody [NB100-137]

Knockdown Validated: HIF Prolyl Hydroxylases Antibody Pack [NB100-903PHD] - 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. EGLN3/PHD3 Antibody [NB100-139]

Immunohistochemistry: HIF Prolyl Hydroxylases Antibody Pack [NB100-903PHD] - Immunofluorescence of wounded tissue and normal unwounded skin. Representative images for immunofluorescent staining of PHD-2 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. EGLN1/PHD2 Antibody [NB100-2219]

Western Blot: HIF Prolyl Hydroxylases Antibody Pack [NB100-903PHD] - Whole cell protein from MEF cells 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 2 ug/mL anti-EGLN3/PHD3 in 1% milk, and detected with an anti-rabbit HRP secondary antibody using chemiluminescence. EGLN3/PHD3 Antibody [NB100-303]

Immunocytochemistry: HIF Prolyl Hydroxylases Antibody Pack [NB100-903PHD] - EGLN1/PHD2 Antibody [NB100-137] - Staining of endogenous PHD2 in U2OS cells. ICC image submitted by a verified customer review. EGLN1/PHD2 Antibody [NB100-137]

Immunohistochemistry: HIF Prolyl Hydroxylases Antibody Pack [NB100-903PHD] - PHD3 was detected in immersion fixed paraffin-embedded sections of human heart using Rabbit Anti-Human EGLN3 polyclonal Antibody (NB100-303) at 5 ug/mL for 1 hour at room temperature followed by incubation with the Anti-Rabbit IgG VisUCyte(TM) HRP Polymer Antibody (VC003). Tissue was stained using DAB (brown) and counterstained with hematoxylin (blue). Specific staining was localized to nuclei. EGLN3/PHD3 Antibody [NB100-303]

Flow Cytometry: HIF Prolyl Hydroxylases Antibody Pack [NB100-903PHD] - Detection of PHD2. 1 million Jurkat cells were fixed, permeabilized, and stained with 3.0 ug/mL anti-PHD2 NB100-137 in a 150 uL reaction. Isotype control (black), anti-MLL1 (red).

Western Blot: HIF Prolyl Hydroxylases Antibody Pack [NB100-903PHD] - Detection of Human PHD2 by Western blot. Samples: Recombinant epitope-tagged PHD1, PHD2 or PHD3 (10 ng/lane) or whole cell lysate from HeLa cells, stained with rabbit anti-PHD2 used at 1 ug/mL. EGLN1/PHD2 Antibody [NB100-138]

Western Blot: HIF Prolyl Hydroxylases Antibody Pack [NB100-903PHD] - Whole cell lysate (50 ug) from HeLa and 293T cells prepared using NETN lysis buffer. Antibody: Affinity purified rabbit anti-PHD4 antibody used for WB at 0.4 ug/mL. Detection: Chemiluminescence with an exposure time of 30 seconds. PHD4/HIF Prolyl Hydroxylase 4 Antibody [NB100-295]

Western Blot: HIF Prolyl Hydroxylases Antibody Pack [NB100-903PHD] - Analysis of PHD2 expression in human prostate cancer cell lines. WB image submitted by a verified customer review. EGLN1/PHD2 Antibody [NB100-137]

Immunohistochemistry: HIF Prolyl Hydroxylases Antibody Pack [NB100-903PHD] - Analysis of a FFPE mouse lung section using 1:200 dilution of EGLN1/PHD2 antibody. The staining was developed using HRP conjugated anti-rabbit secondary antibody and DAB reagent. The antibody generated a specific staining in the cytoplasm and nuclei of alveolar as well as bronchiolar epithelial cells. Cytoplasmic staining was observed in almost all cells while the nuclear positivity was seen in a subset of cells only. EGLN1/PHD2 Antibody [NB100-2219]

Immunohistochemistry: HIF Prolyl Hydroxylases Antibody Pack [NB100-903PHD] - 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. EGLN3/PHD3 Antibody [NB100-139]

Knockdown Validated: HIF Prolyl Hydroxylases Antibody Pack [NB100-903PHD] - In vitro analysis of PHD-2 knockout and protein quantification. Western blot data for PHD-2 knockout in fibroblasts of heterozygous Col1 alpha2-Cre-ER/homozygous floxed PHD-2 mice compared to wild type mice (*p<0.05). D') Western blot data for PHD-2 knockout in keratinocytes of heterozygous K14-Cre/homozygous floxed PHD-2 mice compared to wild type mice (*p<0.05). Image collected and cropped by CiteAb from the following publication (//doi.org/10.1371/journal.pone.0093373), licensed under a CC-BY license. EGLN1/PHD2 Antibody [NB100-2219]

Product Discontinued

HIF Prolyl Hydroxylases Antibody Pack Summary

• Description: This pack contains 1 vial each of: NB100-2219SS (0.025 mL), NB100-303SS (0.025 mL), NB100-139SS (0.025 mL), NB100-138SS (0.025 mL), NB100-295SS (0.025 mL), NB100-137SS (0.025 mL).
• Immunogen: The exact amino acid range is proprietary. General Immunogen area:
  NB100-137 (LocusLink - 54583 Region - N-Terminus),
  NB100-138 (LocusLink - 54583 Region - C-Terminus),
  NB100-139 (LocusLink - 112399 Region - Internal),
  NB100-295 (LocusLink - 54681 Region - C-terminus),
  NB100-303 (LocusLink - 112399 Region - C-terminus),
  NB100-2219 (mouse amino acid range 300-400).
• Specificity: See individual datasheets.
• Host: Rabbit
• Gene: EGLN1
• Purity: Immunogen affinity purified

Applications/Dilutions

• Dilutions:
  • Flow Cytometry
  • Immunocytochemistry/ Immunofluorescence
  • Immunohistochemistry
  • Immunohistochemistry-Paraffin
  • Knockdown Validated
  • Simple Western
  • Western Blot
• Application Notes: These antibodies can be used for Western blot at a dilution range of 1:500 - 1:1,000. Other applications are not confirmed.
  See individual datasheets for validated applications.
• Control:

  EGLN1/PHD2 Overexpression Lysate

Reactivity Notes

All: Homo sapiens.
NB100-137 does not react with mouse.
NB100-138 does not react with mouse and mixed results in rat.
NB100-139 does not react with mouse but reacts with rat.
NB100-295 does not react with mouse and is unknown in rat.
NB100-303 reacts with mouse.
Other species have not been tested.

Packaging, Storage & Formulations

• Storage: Store at 4C. Do not freeze.
• Preservative: 0.01% Sodium Azide
• Concentration: Concentration of individual antibodies may be found on the vial label. If unlisted please contact technical services.
• Purity: Immunogen affinity purified

Alternate Names for HIF Prolyl Hydroxylases Antibody Pack

• C1orf12HPH2
• EC 1.14.11
• EC 1.14.11.-
• ECYT3
• egl nine homolog 1 (C. elegans)
• egl nine homolog 1
• egl nine-like protein 1
• HIF prolyl hydroxylase 2
• HIF-PH2
• HIFPH2EGL nine (C.elegans) homolog 1
• HIF-prolyl hydroxylase 2
• HPH-2
• Hypoxia-inducible factor prolyl hydroxylase 2
• PHD2SM20
• Prolyl hydroxylase domain-containing protein 2
• SM-20
• zinc finger MYND domain-containing protein 6
• ZMYND6DKFZp761F179

Background

Under normoxic conditions, the alpha subunits of the transcriptional complex HIF are degraded by proteasomal degradation via the von Hippel-Lindau ubiquitination complex. The HIF Prolyl Hydroxylases (HPHs) are four enzymes that are involved in this degradation pathway. They catalyze the hydroxylation of HIF-1 alpha and target it for degradation. Under hypoxic conditions, HIF Prolyl Hydroxylase is inhibited, allowing the HIF complex to form and signal Hypoxia Response Elements. HPHs are sometimes referred to as PHDs.

Limitations

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

Publications for HIF Prolyl Hydroxylases Antibody Pack (NB100-903PHD)(15)

Publications using NB100-903PHD

• Siddiq A, Aminova LR, Troy CM et al. Selective Inhibition of Hypoxia-Inducible Factor (HIF) Prolyl-Hydroxylase 1 Mediates Neuroprotection against Normoxic Oxidative Death via HIF- CREB-Independent Pathways. J Neurosci;29(27):8828-8838. 2009-01-01 [PMID: 19587290]
• Loinard C, Ginouves A, Vilar J et al. Inhibition of prolyl hydroxylase domain proteins promotes therapeutic revascularization. Circulation 2009-07-01 [PMID: 19546390]
• Schultz K, Murthy V, Tatro JB et al. Prolyl hydroxylase 2 deficiency limits proliferation of vascular smooth muscle cells by hypoxia-inducible factor-1{alpha}-dependent mechanisms. Am J Physiol Lung Cell Mol Physiol;296(6):L921-927. 2009-01-01 [PMID: 19304911]
• Mikhaylova, O, Ignacak, M et al. The von Hippel-Lindau tumor suppressor protein and Egl-9-Type proline hydroxylases regulate the large subunit of RNA polymerase II in response to oxidative stress. Mol Cell Biol;28(8):2701-17. 2008-04-01 [PMID: 18285459]
• Chang, E et al. Age decreases endothelial progenitor cell recruitment through decreases in hypoxia-inducible factor 1alpha stabilization during ischemia. Circulation;116(24):2818-29. 2007-12-11 [PMID: 18040029]
• Bex, C et al. A yeast two-hybrid system reconstituting substrate recognition of the von Hippel-Lindau tumor suppressor protein. Nucleic Acids Res;35(21):e142. 2007-01-01 [PMID: 17986458]
• Baek, JH et al. Spermidine/spermine N(1)-acetyltransferase-1 binds to hypoxia-inducible factor-1alpha (HIF-1alpha) and RACK1 and promotes ubiquitination and degradation of HIF-1alpha. J. Biol. Chem. 282(46): 33358-33366. 2007-01-01 [PMID: 17875644]
• Colla S, Tagliaferri S, Morandi F, Lunghi P, Donofrio G et al. The new tumor-suppressor gene inhibitor of growth family member 4 (ING4) regulates the production of proangiogenic molecules by myeloma cells and suppresses hypoxia-inducible factor-1 alpha (HIF-1alpha) activity: involvement in myeloma-induced angiogenesis. Blood;110(13):4464-75. 2007-12-15 [PMID: 17848618]
• Baek, JH et al. Spermidine/spermine-N1-acetyltransferase 2 is an essential component of the ubiquitin ligase complex that regulates hypoxia-inducible factor 1alpha. J. Biol. Chem.82(32): 23572-23580. 2007-01-01 [PMID: 17558023]
• Choi, Y et al. AKAP12 regulates human blood-retinal barrier formation by downregulation of hypoxia-inducible factor-1alpha. J Neurosci;27(16):4472-81. 2007-04-18 [PMID: 17442832]
• Grover, TR et al. Hypoxia-inducible factors HIF-1alpha and HIF-2alpha are decreased in an experimental model of severe respiratory distress syndrome in preterm lambs. Am J Physiol Lung Cell Mol Physiol;292(6):L1345-51. 2007-06-01 [PMID: 17307811]
• Kondo, S et al. EBV latent membrane protein 1 up-regulates hypoxia-inducible factor 1alpha through Siah1-mediated down-regulation of prolyl hydroxylases 1 and 3 in nasopharyngeal epithelial cells. Cancer Res;66(20):9870-7. 2006-10-15 [PMID: 17047048]
• McMahon, S et al. PROTEIN SYNTHESIS, POST-TRANSLATION MODIFICATION, DEGRADATION: TGF beta 1 induces HIF-1 stabilization through selective inhition of PHD2 expression. J Biol Chem, 10.1074/jbc.M604507200. 2006-01-01 [PMID: 16815840]
• Stiehl, DP et al. Increased prolyl 4-hydroxylase domain proteins compensate for decreased oxygen levels. Evidence for an autoregulatory oxygen-sensing system. J Biol Chem;281(33):23482-91. 2006-08-18 [PMID: 16790428]
• Lu H et al. Reversible inactivation of HIF-1 prolyl hydroxylases allows cell metabolism to control basal HIF-1. J Biol Chem;280(51):41928-39. 2005-12-23 [PMID: 16223732]

Bioinformatics

• Gene Symbol: EGLN1