FIG. 3.

FLIP blocks the association of Bid and Bax and inhibits Bax-induced cell death in the absence of Bid. Wild-type (A) or Bid knockout (Bid^−/−) (B) 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 normal medium. Two days later, cells were exposed to hypoxia (95% N₂, 5% CO₂) for 24 h and then restored to normoxic culture conditions (95% air, 5% CO₂). At the indicated times, cell lysates were subjected to immunoprecipitation (IP) with antibody 6A7 followed by immunoblotting (IB) to detect Bid (A) or Bax (B). For the toxicity assay, MLEC (Bid^−/−) were treated with H/R as described above. At the indicated reoxygenation time, 200 μl of supernatant medium was removed for LDH assays as described in Materials and Methods (C). The data represent averages of the results from two independent experiments with each sample in triplicate (n = 3). Data from adeno-FLIP-infected cells were compared with control (adeno-lacZ-infected) cells at each time point using Student's t test (*P < 0.05). O.D., optical density.