Primary antibodies against hif 1α

Cells were cross-linked with 1% formaldehyde for 10 min and quenched in 0.125 M glycine. Chromatin was sheared by sonication using a Covaris sonicator (settings: Power (W): 150, Duty Factor: 5%, Cycles: 200 Treatment time: 420 sec (7 min)). Sonicated lysates were precleared with salmon sperm DNA/protein A agarose slurry (Millipore). IgG (Santa Cruz) or primary antibodies against HIF-1α (Santa Cruz), HIF-2α (Novus Biologicals), or HIF-1β (Novus Biologicals) were added and incubated overnight with precleared lysates. The following day, salmon sperm DNA/protein A agarose beads were added for 4 h at 37°C. The agarose beads were collected and washed sequentially with: low- and high-salt immune complex wash buffers; LiCl immune complex wash buffer; and twice with TE buffer. The DNA was eluted from the agarose gel in 1% SDS/0.1 M NaHCO₃ and crosslinks were reversed by addition of NaCl to a final concentration of 0.2 M. Proteinase K was added to degrade protein in the lysate. DNA was recovered by phenol-chloroform extraction followed by ethanol precipitation, treated with RNase, and analyzed by qPCR. Fold enrichment was calculated based on the cycle threshold (Ct) as 2^−Δ(ΔC_t⁾, where ΔC_t = C_t,IP − C_t,Input and Δ(ΔC_t) = ΔC_t,antibody − ΔC_t,IgG.