Bortezomib enhances FIH binding to HIF-1α, and then blocks p300 recruitment by HIF-1α. (A) Bortezomib stimulated the interaction between HIF-1α and FIH. HEK293 cells were cotransfected with FIH and HIF-1α plasmids and then incubated with bortezomib (0.1 and 1 nM) or MG132 (10 μM) under hypoxic conditions for 8 hours. Lysates were prepared and immunoprecipitated using anti–HIF-1α, anti-FIH, or nonimmunized rabbit serum (pre-igG). Coimmunoprecipitated FIH or HIF-1α was identified by Western blotting. (B) FIH knock-down rescues HIF-1α–p300 binding which is inhibited by bortezomib. HEK293 cells were cotransfected with HIF-1α (2 μg) and p300 (1 μg) plasmids, and incubated under normoxic or hypoxic conditions with 1 nM bortezomib for 8 hours. p300 was immunoprecipitated with anti-p300 antiserum and protein G/A beads, and precipitated p300 and coprecipitated HIF-1α was identified by Western blotting.