Cell Signaling Technology
XP Monoclonal Antibody

Product Pathways - Translational Control

Hydroxy-HIF-1α (Pro564) (D43B5) XP® Rabbit mAb #3434

Applications Reactivity Sensitivity MW (kDa) Isotype
W IP IF-IC H (M) (R) (Mk) (C) (X) (Z) (Pg) Endogenous 120 Rabbit IgG

Applications Key:  W=Western Blotting  IP=Immunoprecipitation  IF-IC=Immunofluorescence (Immunocytochemistry)
Reactivity Key:  H=Human  M=Mouse  R=Rat  Mk=Monkey  C=Chicken  X=Xenopus  Z=Zebrafish  Pg=Pig
Species cross-reactivity is determined by western blot. Species enclosed in parentheses are predicted to react based on 100% sequence homology.

Protocols

Specificity / Sensitivity

Hydroxy-HIF-1α (Pro564) (D43B5) XP® Rabbit mAb detects endogenous levels of HIF-1α only when hydroxylated at Pro564. This antibody may cross react with other overexpressed proline hydroxylated proteins.

Source / Purification

Monoclonal antibody is produced by immunizing animals with a synthetic hydroxypeptide corresponding to residues surrounding Pro564 of human HIF-1α.

Western Blotting

Western Blotting

Western blot analysis of extracts from HeLa cells, treated with either 10 μM of MG132 (to accumulate hydroxylated HIF-1α) or 10 µM MG132 and 1 mM DMOG (to accumulate nonhyroxylated HIF-1α), using Hydroxy-HIF-1α (Pro564) (D43B5) XP® Rabbit mAb (upper) or total HIF-1α Antibody #3716 (lower).

IF-IC

IF-IC

Confocal immunofluorescent analysis of HeLa cells, treated with either 10 μM MG132 (left) or 10 μM MG132 and 1 mM DMOG (right), using Hydroxy-HIF-1α (Pro564) (D43B5) XP® Rabbit mAb (green). Actin filaments have been labeled using DY-554 phalloidin (red).

Background

Hypoxia-inducible factor 1 (HIF1) is a heterodimeric transcription factor that plays a critical role in the cellular response to hypoxia (1). The HIF1 complex consists of two subunits, HIF-1α and HIF-1β, which are basic helix-loop-helix proteins of the PAS (Per, ARNT, Sim) family (2). HIF1 regulates the transcription of a broad range of genes that facilitate responses to the hypoxic environment, including genes regulating angiogenesis, erythropoiesis, cell cycle, metabolism and apoptosis. The widely expressed HIF-1α is typically degraded rapidly in normoxic cells by the ubiquitin/proteasomal pathway. Under normoxic conditions, HIF-1α is proline hydroxylated leading to a conformational change that promotes binding to the von Hippel Lindau protein (VLH) E3 ligase complex; ubiquitination and proteasomal degradation follows (3,4). Both hypoxic conditions and chemical hydroxylase inhibitors (such as desferrioxamine and cobalt) inhibit HIF-1α degradation and lead to its stabilization. In addition, HIF-1α can be induced in an oxygen-independent manner by various cytokines through the PI3K-AKT-mTOR pathway (5-7).HIF-1β is also known as AhR nuclear translocator (ARNT) due to its ability to partner with the aryl hydrocarbon receptor (AhR) to form a heterodimeric transcription factor complex (8). Together with AhR, HIF-1β plays an important role in xenobiotics metabolism (8). In addition, a chromosomal translocation leading to a TEL-ARNT fusion protein is associated with acute myeloblastic leukemia (9). Studies also found that ARNT/HIF-1β expression levels decrease significantly in pancreatic islets from patients with type 2 diabetes, suggesting that HIF-1β plays an important role in pancreatic β-cell function (10).

Two critical prolines in HIF-1α (Pro564 and Pro402) can be hydroxylated by proline hydroxylase under normoxia conditions. Hydroxylation of HIF-1α leads to its binding to VHL and ubiquitin mediated degradation (3,11,12).

  1. Sharp, F.R. and Bernaudin, M. (2004) Nat Rev Neurosci 5, 437-48.
  2. Wang, G.L. et al. (1995) Proc Natl Acad Sci U S A 92, 5510-4.
  3. Jaakkola, P. et al. (2001) Science 292, 468-72.
  4. Maxwell, P.H. et al. (1999) Nature 399, 271-5.
  5. Fukuda, R. et al. (2002) J Biol Chem 277, 38205-11.
  6. Jiang, B.H. et al. (2001) Cell Growth Differ 12, 363-9.
  7. Laughner, E. et al. (2001) Mol Cell Biol 21, 3995-4004.
  8. Walisser, J.A. et al. (2004) Proc Natl Acad Sci U S A 101, 16677-82.
  9. Salomon-Nguyen, F. et al. (2000) Proc Natl Acad Sci U S A 97, 6757-62.
  10. Gunton, J.E. et al. (2005) Cell 122, 337-49.
  11. Ivan, M. et al. (2001) Science 292, 464-8.
  12. Masson, N. et al. (2001) EMBO J 20, 5197-206.

Application References

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