Amphotericin B blocks p300 recruitment by binding FIH to CAD. (A) Coimmunoprecipitation assay for FIH-CAD binding. HEK293 cells were cotransfected with pHA-FIH and either pGal4-CAD or pGal4, and incubated with sodium deoxycholate (-) or AmB under hypoxic conditions for 16 hours. Lysates were prepared and immunoprecipitations were performed using anti-Gal4 antibody. The coimmunoprecipitation of HA-FIH and Gal4-CAD was identified by Western blotting using anti-HA antibody (bottom panel). Protein quantities in the samples were verified using anti-Gal4 and anti-HA antibodies (top and middle panels). The data shown are representative of 3 separate experiments. (B) Mammalian 2-hybrid assay for CAD-p300 binding. Hep3B (left panel) and HEK293 (right panel) cells were cotransfected with 1 μg of a Gal4-luciferase reporter, 1 μg pGal4-CAD, and 500 ng p(His)VP16-C/H1. After a stabilizing period of 48 hours, cells were incubated under either normoxic or hypoxic conditions, in the absence or presence of AmB (5 μg/mL) for 16 hours, and then lysed to determine luciferase and β-gal activities. The results shown are relative values versus the normoxic control and are plotted as the means ± SD of 8 experiments.