The HIF (hypoxia-inducible factor) transcription factor is a member of the basic-helix-loop-helix (bHLH) family of transcription factors and plays an important role in maintaining cellular oxygen homeostasis.^1,2 HIF-1α has emerged as an important drug target in breast and prostate cancer, cardiovascular disease, and ischemia.^3,4,5 Cayman’s HIF-1α Transcription Factor Assay is a non-radioactive, sensitive method for detecting specific transcription factor DNA binding activity in nuclear extracts and whole cell lysate. A 96-well enzyme-linked immunosorbent assay (ELISA) replaces the cumbersome radioactive electrophoretic mobility shift assay (EMSA). A specific double stranded DNA (dsDNA) consensus sequence containing the HIF-1α response element is immobilized to the wells of a 96-well plate. HIF-1α contained in a nuclear extract or whole cell lysates, binds specifically to the HIF-1α response element. The HIF transcription factor complex is detected by addition of a specific primary antibody directed against HIF-1α. A secondary antibody conjugated to HRP is added to provide a sensitive colorimetric readout at 450 nm.

1 Wang, G.L., Jiang, B., Rue, E.A., et al. Hypoxia-inducible factor 1 is a basic-helix-loop-helix-PAS heterodimer regulated by cellular O₂ tension. Proc Natl Acad Sci USA 92 5510-5514 (1995).

2 Safran, M., and Kaelin, W.G. HIF hydroxylation and the mammalian oxygen-sensing pathway. J Clin Invest 111 779-783 (2003).

3 Rapisarda, A., Uranchimeg, B., Scudiero, D.A., et al. Identification of small molecule inhibitors of hypoxia-inducible factor 1 transcriptional activation pathway. Cancer Res 62 4316-4324 (2002).

4 William, C., Masson, N., Tian, Y., et al. Peptide blockade of HIFα degradation modulates cellular metabolism and angiogenesis. Proc Natl Acad Sci USA 99(16) 10423-10428 (2002).

5 Welsh, S., Williams, R., Kirkpatrick, L., et al. Antitumor activity and pharmacodynamic properties of PX-478, an inhibitor of hypoxia-inducible factor-1α. Mol Cancer Ther 3(3) 233-244 (2004).

Wang, G.L., Jiang, B., Rue, E.A., et al. Hypoxia-inducible factor 1 is a basic-helix-loop-helix-PAS heterodimer regulated by cellular O₂ tension. Proc Natl Acad Sci USA 92 5510-5514 (1995).

Kabra, P.M. Clinical analysis of individual steroids by column liquid chromatography. J Chromatogr 429 155-176 (1988).

William, C., Masson, N., Tian, Y., et al. Peptide blockade of HIFα degradation modulates cellular metabolism and angiogenesis. Proc Natl Acad Sci USA 99(16) 10423-10428 (2002).

Welsh, S., Williams, R., Kirkpatrick, L., et al. Antitumor activity and pharmacodynamic properties of PX-478, an inhibitor of hypoxia-inducible factor-1α. Mol Cancer Ther 3(3) 233-244 (2004).

Rapisarda, A., Uranchimeg, B., Scudiero, D.A., et al. Identification of small molecule inhibitors of hypoxia-inducible factor 1 transcriptional activation pathway. Cancer Res 62 4316-4324 (2002).

Kimbro, K.S., and Simons, J.W. Hypoxia-inducible factor-1 in human breast and prostate cancer. Endocr Relat Cancer 13 739-749 (2006).

Safran, M., and Kaelin, W.G. HIF hydroxylation and the mammalian oxygen-sensing pathway. J Clin Invest 111 779-783 (2003).

Koukourakis, M.I., Giatromanolaki, A., Skarlatos, J., et al. Hypoxia inducible factor (HIF-1α and HIF-2α) expression in early esophageal cancer and response to photodynamic therapy and radiotherapy. Cancer Res 61 1830-1832 (2001).

Turner, K.J., Moore, J.W., Jones, A., et al. Expression of hypoxia-inducible factors in human renal cancer: Relationship to angiogenesis and to the von Hippel-Lindau gene mutation. Cancer Res 62 2957-2961 (2002).

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