Fig. 4.

Cells and mitochondria lacking multidomain Bax and Bak are resistant to heat. (A) WT or DKO MEFs were incubated for 1 h at the indicated temperatures, followed by incubation at 37°C for an additional 5 h. Samples were incubated with annexinV-FITC and analyzed for apoptosis by flow cytometry. (B) Cells were heated as in A, harvested, and treated with digitonin. The cytosol and mitochondria (pellet) were analyzed by Western blot for the presence of cytochrome c and VDAC. A nonspecific (ns) band is displayed as a loading control. (C) Mitochondria enriched from WT (WT mito) or DKO (DKO mito) MEFs were heated at 43°C for the indicated times, followed by incubation for 1 h at 37°C. Cytochrome c release from the mitochondria (pellet) to supernatant was assayed by Western blot. (D) DKO mitochondria were enriched and incubated with 50 nM FL Bax on ice or at 43°C for 1 h, followed by incubation for 1 h at 37°C. Samples were assayed as in C.