FIG. 2.

FLIP inhibits Bax activation induced by hypoxia/reoxygenation in MLEC. MLEC were transfected with Bax siRNA or control siRNA for 36 h, exposed to hypoxia (95% N₂, 5% CO₂) for 24 h, and then restored to normal culture conditions (95% air, 5% CO₂). At the indicated reoxygenation time, 200 μl of supernatant medium was removed for LDH assays as described in Materials and Methods (A). MLEC at 30% confluence were cultured in serum-free media in the presence of 10⁶ CPU/ml of adeno-FLIP or adeno-lacZ for 3 h and then restored to complete growth medium. Two days later, the cells were exposed to hypoxia (95% N₂, 5% CO₂) for 24 h and then restored to normal culture conditions (95% air, 5% CO₂) for the indicated reoxygenation times. Total lysates (B) or fractions consisting of cytosol or mitochondria (C) were subjected to immunoprecipitation with antibody 6A7 to detect Bax (immunoblotting [IB]). O.D., optical density.