Figure 5. BCL-xL blocks MOMP sequentially through MODE 1 and MODE 2.

(A) MHM fractions extracted from C57BL/6 mouse liver were incubated for 1 hr at 37°C with 100 nM full length recombinant BCL-xL and the indicated concentrations of cleaved recombinant BID (n/cBID). MHM were then incubated with calpain (20 nM) in presence of CaCl₂ (0.5 mM) for 1hr at room temperature. BAK cleavage was monitored by Western blot using anti-BAK-G23 antibody. Full-length BAK (BAK FL) and two cleavage fragments (BH4 Cl and BH3 Cl) are indicated with arrows. Supernatants (S) and mitochondrial pellets (P) were also analyzed for cytochrome c by Western blot. (B) HeLa cells stably over-expressing BCL-xL or an empty vector were transiently transfected with the indicated amount of tBID for 24 hr in presence of Q-VD-OPH (20 μM), permeabilized with digitonin (200 μg/ml) and incubated with calpain (20 nM) in presence of CaCl₂ (0.5 mM) for 30 min at room temperature. BAK cleavage was monitored by Western blot using anti-BAK-G23 antibody. Presence of cytochrome c was analyzed in pellet fractions (P) only as cytosolic components were lost during digitonin permeabilization. (C) HeLa cells stably over-expressing BCL-xL or an empty vector were exposed to increasing UV intensity (0, 2.5, 5, 10, 20 and 40 mJ/cm²) in presence of Q-VD-OPH (20 μM). After 16 hr, cells were treated as in (B) for calpain proteolysis (upper panel) or monitored for cytochrome c release (middle panel). Apoptosis was assessed on intact cells by Annexin V staining and flow cytometry analysis (lower panel). Error bars represent standard deviation from triplicate data. (D) MHM fractions from C57BL/6 mouse liver were incubated for 1 hr at 37°C with full-length recombinant BCL-xL (100 nM) and n/cBID (10 nM). Reactions were then de-repressed with the indicated concentrations of BAD-BH3 peptide or ABT-737 for 1 hr at 37°C and treated as in (C) for calpain proteolysis (upper panel) or monitored for cytochrome c release (lower panel).

(See also Figure S3)