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

Hypoxia Activation IHC Antibody Sampler Kit #43065

    Product Information

    Kit Usage Information

    Protocols

    Product Description

    The Hypoxia Activation IHC Antibody Sampler Kit provides an economical means of detecting select components involved in the regulation of HIF-1α, select components regulated by HIF-1α, and HIF-1β/ARNT protein in formalin-fixed, paraffin-embedded tissue samples.

    Specificity / Sensitivity

    Each antibody in the Hypoxia Activation IHC Antibody Sampler Kit detects endogenous levels of its target protein. HIF-1α (E1V6A) Rabbit mAb does not cross-react with HIF-2α protein. Non-specific staining of skeletal and cardiac muscle has been observed by immunohistochemistry using VHL (E3X9K) Rabbit mAb. p300 (D8Z4E) Rabbit mAb does not cross-react with CBP protein. GSK-3β (D5C5Z) XP® Rabbit mAb does not cross-react with GSK-3α protein. PKM2 (D78A4) XP® Rabbit mAb does not cross-react with PKM1 protein. Glut1 (E4S6I) Rabbit mAb does not cross-react with Glut2, Glut3, or Glut4 protein.

    Source / Purification

    Monoclonal antibodies are produced by immunizing animals with synthetic peptides corresponding to residues surrounding Ala475 of human HIF-1α protein, Ile479 of human HIF-1β/ARNT protein, Ser406 of human PKM2 protein, near the carboxy terminus of human p300 protein and human Glut1 protein, and corresponding to the sequence of human LDHA protein.
    Monoclonal antibodies are also produced by immunizing animals with recombinant proteins specific to human VHL protein, representing the central region of human SirT1 protein, and specific to the carboxy terminus of human GSK-3β 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). CBP (CREB-binding protein) and p300 are highly conserved and functionally related transcriptional co-activators that associate with transcriptional regulators and signaling molecules, integrating multiple signal transduction pathways with the transcriptional machinery (11,12). CBP/p300 also contain histone acetyltransferase (HAT) activity, allowing them to acetylate histones and other proteins (12). The Silent Information Regulator (SIR2) family of genes is a highly conserved group of genes that encode nicotinamide adenine dinucleotide (NAD)-dependent protein deacetylases, also known as class III histone deacetylases. The first discovered and best characterized of these genes is Saccharomyces cerevisiae SIR2, which is involved in silencing of mating type loci, telomere maintenance, DNA damage response, and cell aging (13). SirT1, the mammalian ortholog of Sir2, is a nuclear protein implicated in the regulation of many cellular processes, including apoptosis, cellular senescence, endocrine signaling, glucose homeostasis, aging, and longevity. Targets of SirT1 include acetylated p53 (14,15), p300 (16), Ku70 (17), forkhead (FoxO) transcription factors (17,18), PPARγ (19), and the PPARγ coactivator-1α (PGC-1α) protein (20). Glycogen synthase kinase-3 (GSK-3) was initially identified as an enzyme that regulates glycogen synthesis in response to insulin (21). GSK-3 is a ubiquitously expressed serine/threonine protein kinase that phosphorylates and inactivates glycogen synthase. GSK-3 is a critical downstream element of the PI3K/Akt cell survival pathway whose activity can be inhibited by Akt-mediated phosphorylation at Ser21 of GSK-3α and Ser9 of GSK-3β (22,23). Pyruvate kinase is a glycolytic enzyme that catalyzes the conversion of phosphoenolpyruvate to pyruvate. In mammals, the M2 isoform (PKM2) is expressed during embryonic development (24). Lactate dehydrogenase (LDH) catalyzes the interconversion of pyruvate and NADH to lactate and NAD+. The major form of LDH found in muscle cells is the A (LDHA) isozyme (25). Glucose transporter 1 (Glut1, SLC2A1) is a widely expressed transport protein that transports a number of different aldose sugars into cells (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. Goodman, R.H. and Smolik, S. (2000) Genes Dev 14, 1553-77.
    12. Chan, H.M. and La Thangue, N.B. (2001) J Cell Sci 114, 2363-73.
    13. Guarente, L. (1999) Nat Genet 23, 281-5.
    14. Vaziri, H. et al. (2001) Cell 107, 149-59.
    15. Luo, J. et al. (2001) Cell 107, 137-48.
    16. Bouras, T. et al. (2005) J Biol Chem 280, 10264-76.
    17. Brunet, A. et al. (2004) Science 303, 2011-5.
    18. Motta, M.C. et al. (2004) Cell 116, 551-63.
    19. Picard, F. et al. (2004) Nature 429, 771-6.
    20. Rodgers, J.T. et al. (2005) Nature 434, 113-8.
    21. Welsh, G.I. et al. (1996) Trends Cell Biol 6, 274-9.
    22. Srivastava, A.K. and Pandey, S.K. (1998) Mol Cell Biochem 182, 135-41.
    23. Cross, D.A. et al. (1995) Nature 378, 785-9.
    24. Christofk, H.R. et al. (2008) Nature 452, 230-3.
    25. Semenza, G.L. et al. (1996) J Biol Chem 271, 32529-37.
    26. Ferrer, C.M. et al. (2014) Mol Cell 54, 820-31.
    27. Deng, D. et al. (2014) Nature 510, 121-5.

    Database Links

    UniProt ID: Q16665 , P00338 , P11166 , P40337 , P27540 , Q09472 , Q96EB6 , P14618 , P49841


    Entrez-Gene Id: 3091 , 3939 , 6513 , 7428 , 405 , 2033 , 23411 , 5315 , 2932


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