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PDP - Template Name: Antibody Sampler Kit
PDP - Template ID: *******4a3ef3a
  1. Products /
  2. Primary Antibodies /
  3. Hypoxia Pathway Antibody Sampler Kit

Hypoxia Pathway Antibody Sampler Kit #15792

    Product Information

    Kit Usage Information

    Protocols

    Product Description

    The Hypoxia Pathway Antibody Sampler Kit provides an economical means to investigate select proteins involved in the hypoxia pathway. The kit contains enough primary antibodies to perform two western blot experiments with each primary antibody.

    Specificity / Sensitivity

    HIF-1α (D1S7W) XP® Rabbit mAb recognizes endogenous levels of total HIF-1α protein. This antibody does not cross-react with HIF-2α protein. 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. HIF-1β/ARNT (D28F3) XP® Rabbit mAb detects endogenous levels of total HIF-1β/ARNT protein. HIF-2α (D6T8V) Rabbit mAb recognizes endogenous levels of total HIF-2α protein. This antibody does not cross-react with HIF-1α protein. FIH (D19B3) Rabbit mAb detects endogenous levels of total FIH protein. PHD-2/Egln1 (D31E11) Rabbit mAb detects endogenous levels of total PHD-2/Egln1 protein. VHL Antibody recognizes endogenous levels of total VHL protein.

    Source / Purification

    Monoclonal antibodies are produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Leu478 of human HIF-1α protein, Gly688 of human HIF-2α protein, Tyr35 of human FIH protein, and Val226 of human PHD-2/Egln1 protein. Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to the sequence around Ile479 of human HIF-1β/ARNT protein. Monoclonal antibody is produced by immunizing animals with a synthetic hydroxypeptide corresponding to residues surrounding Pro564 of human HIF-1α. Polyclonal antibodies are produced by immunizing animals with a synthetic peptide corresponding to residues near the carboxy terminus of human VHL protein.

    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 (VHL) 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).

    Hypoxia-inducible factor (HIF) is essential for the cellular response to hypoxia (11,12). There are several isoforms of the HIF α subunit. Studies have found that HIF-1α and HIF-2α expression is increased in some human cancers. HIF-1α has both pro- and anti-proliferative activities, whereas HIF-2α does not possess anti-proliferative activity (12). Therefore, HIF-2α likely plays an important role in tumorigenesis (12,13).

    FIH (Factor inhibiting HIF-1, HIF asparagine hydroxylase) is a dioxygen-dependent asparaginyl hydroxylase that modifies target protein function by hydroxylating target protein asparagine residues (14-16). HIF, a transcriptional activator involved in control of cell cycle in response to hypoxic conditions, is an important target for FIH regulation. FIH functions as an oxygen sensor that regulates HIF function by hydroxylating at Asn803 in the carboxy-terminal transactivation domain (CAD) of HIF (17,18). During normoxia, FIH uses cellular oxygen to hydroxylate HIF-1 and prevent interaction of HIF-1 with transcriptional coactivators, including the CBP/p300-interacting transactivator. Under hypoxic conditions, FIH remains inactive and does not inhibit HIF, allowing the activator to regulate transcription of genes in response to low oxygen conditions (17-19). FIH activity is regulated through interaction with proteins, including Siah-1, which targets FIH for proteasomal degradation (20). The Cut-like homeodomain protein CDP can bind the FIH promoter region to regulate FIH expression at the transcriptional level (21). Phosphorylation of HIF at Thr796 also can prevent FIH hydroxylation on Asn803 (22). Potential FIH substrates also include proteins with ankyrin repeat domains, such as Iκ-B, Notch, and ASB4 (23-25).

    PHD1 (Egln2), PHD-2 (Egln1), and PHD3 (Egln3) are members of the Egln family of proline hydroxylases. They function as oxygen sensors that catalyze the hydroxylation of HIF on prolines 564 and 402, initiating the first step of HIF degradation through the VHL/ubiquitin pathway (26,27).

    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. Kaelin, W.G. (2005) Biochem Biophys Res Commun 338, 627-38.
    12. Toschi, A. et al. (2008) J Biol Chem 283, 34495-9.
    13. Gordan, J.D. and Simon, M.C. (2007) Curr Opin Genet Dev 17, 71-7.
    14. Koivunen, P. et al. (2004) J Biol Chem 279, 9899-904.
    15. Linke, S. et al. (2004) J Biol Chem 279, 14391-7.
    16. Lisy, K. and Peet, D.J. (2008) Cell Death Differ 15, 642-9.
    17. Mahon, P.C. et al. (2001) Genes Dev 15, 2675-86.
    18. Lando, D. et al. (2002) Genes Dev 16, 1466-71.
    19. Lando, D. et al. (2002) Science 295, 858-61.
    20. Fukuba, H. et al. (2007) Biochem Biophys Res Commun 353, 324-9.
    21. Li, J. et al. (2007) Mol Cell Biol 27, 7345-53.
    22. Lancaster, D.E. et al. (2004) Biochem J 383, 429-37.
    23. Ferguson, J.E. et al. (2007) Mol Cell Biol 27, 6407-19.
    24. Cockman, M.E. et al. (2006) Proc Natl Acad Sci U S A 103, 14767-72.
    25. Cockman, M.E. et al. (2009) Mol Cell Proteomics 8, 535-46.
    26. Freeman, R.S. et al. (2003) Mol Cells 16, 1-12.
    27. Villar, D. et al. (2007) Biochem J 408, 231-40.

    Database Links

    UniProt ID: Q16665 , P40337 , P27540 , Q9NWT6 , Q99814 , Q9GZT9


    Entrez-Gene Id: 3091 , 7428 , 405 , 55662 , 2034 , 54583


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