Detection of HIF‑1 alpha -regulated Genes by Chromatin Immunoprecipitation. Mouse primary kidney cells treated with 150 µM CoCl2 for overnight were fixed using formaldehyde, resuspended in lysis buffer, and sonicated to shear chromatin. HIF‑1 alpha/DNA complexes were immunoprecipitated using 5 µg Goat Anti-Human/Mouse/Rat HIF‑1 alpha Antigen Affinity-purified Polyclonal Antibody (Catalog # AF1935) 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 epo promoter was detected by standard PCR.

Detection of Human HIF‑1 alpha by Simple WesternTM. Simple Western lane view shows lysates of A549 human lung carcinoma cell line untreated (-) or treated (+) with Hypoxia (1% O2), loaded at 0.2 mg/mL. A specific band was detected for HIF‑1 alpha at approximately 115 kDa (as indicated) using 5 µg/mL of Goat Anti-Human/Mouse/Rat HIF‑1 alpha Antigen Affinity-purified Polyclonal Antibody (Catalog # AF1935) followed by 1:50 dilution of HRP-conjugated Anti-Goat IgG Secondary Antibody (Catalog # HAF109). This experiment was conducted under reducing conditions and using the12-230 kDa separation system.