FIG. 5.

(A) Mitochondrial cytochrome c release induced by recombinant WT, DM56/61, and tBAD₆₈ BAD proteins. Recombinant WT, double-mutant, and truncated BAD proteins fused to the TAT leader sequence were generated and purified as described in Materials and Methods. Partially purified mitochondria (15 μl) from parental 32Dc13 cells were incubated with a vehicle (lane 1; 250 mM sucrose buffer) or 100 nM WT BAD (lanes 2 and 3), double-mutant BAD (lanes 4 and 5), or truncated BAD (lane 6) in the absence (lanes 2, 4, and 6) or presence (lanes 1, 3, and 5) of 1 μl of recombinant caspase 3 (10 ng) in 30 μl. After 1 h at 30°C, samples were centrifuged at 12,000 × g for 10 min at 4°C. Supernatants (Sup) were subjected to an SDS–4 to 15% gradient polyacrylamide gel and transferred to a nitrocellulose filter. The filter was probed with a monoclonal anti-cytochrome c antibody (α-Cytochrome C) (upper gel). Mitochondrial pellets were lysed, and protein extracts were blotted with an antibody to subunit IV of cytochrome oxidase COX-IV) as a control for equal loadings (middle gel). The same filter was blotted with anti-cytochrome c antibody to monitor the presence of residual protein in the mitochondrial fraction (lower gel). (B) Densitometric analysis of cytochrome c release. The intensities of the cytochrome c band in the supernatants were measured and normalized against that of COX-IV detected in the pellet extracts, used as control (CTRL) of mitochondrial loading. Data are the ratios of the value of each cytochrome c band to that of its COX-IV counterpart. Results are representative of two separate experiments.