|Detection of Human and Rat HIF-2 alpha /EPAS1 by Western Blot. Western blot shows lysates of MCF‑7 human breast cancer cell line and PC‑12 rat adrenal pheochromocytoma cell line untreated (-) or treated (+) with 150 µM CoCl2 for 16 h. PVDF membrane was probed with 1 µg/mL of Human/Rat HIF‑2 alpha /EPAS1 Polyclonal Antibody, followed by HRP-conjugated Anti-Goat IgG Secondary Antibody (Catalog # HAF109). A specific band was detected for HIF‑2 alpha /EPAS1 at approximately 115 kDa (as indicated). This experiment was conducted under reducing conditions and using Immunoblot Buffer Group 1.|
Chromatin Immunoprecipitation (ChIP)
|Detection of HIF‑2 alpha /EPAS1-regulated Genes by Chromatin Immunoprecipitation. BG01V human embryonic stem cells treated with 150 μM CoCl2 overnight were fixed using formaldehyde, resuspended in lysis buffer, and sonicated to shear chromatin. HIF‑2 alpha /EPAS1/DNA complexes were immunoprecipitated using 5 μg Goat Anti-Human/Rat HIF‑2 alpha /EPAS1 Antigen Affinity-purified Polyclonal Antibody (Catalog # AF2886) or control antibody (Catalog # AB-108-C) for 15 minutes in an ultrasonic bath, followed by Biotinylated Anti-Goat IgG Secondary Antibody (Catalog # BAF109). Immunocomplexes were captured using 50 μL of MagCellect Streptavidin Ferrofluid (Catalog # MAG999) and DNA was purified using chelating resin solution. The oct-3/4 promoter was detected by standard PCR.|
The hypoxia-inducible transcription factor 2 alpha (HIF-2 alpha ) is stabilized in hypoxic tissue and, similarly to HIF-1 alpha, complexes with Aryl hydrocarbon receptor nuclear translocator (ARNT). Both the HIF-1 and HIF-2 complexes bind hypoxia-response elements (HREs) in the promoters of many genes involved in adapting to an environment of insufficient oxygen or hypoxia. HIF-1 and HIF-2 do not appear completely redundant, although specific functions are only beginning to be elucidated. Hypoxic tissue environments occur in vascular and pulmonary diseases as well as cancer, which illustrates the potentially broad impact of gene regulation by both HIF-1 alpha and HIF-2 alpha.
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