Fig. 5.
Cytosol from untreated cells buffers the effect of heat on enriched mitochondria and Bax. (A) CE (4 mg/ml) from untreated Jurkat cells was heated with mouse-liver mitochondria at 43°C for 30 or 60 min, followed by incubation for 1 h at 37°C. The mitochondria (pellet) and supernatant were probed for cytochrome c by Western blot. (B) Jurkat CE was immunodepleted with anti-Bcl-x antibody (↓xL) or control antibody (↓Ig), and the presence of Bcl-xL was detected by Western blotting. Each extract (4 mg/ml) was heated with mitochondria at 43°C for 60 min, and cytochrome c release was determined by Western blot. (C) Recombinant BaxΔC (15 μM) was heated at 43°C for 1 h, either alone or with 200 μg/ml WT, mutant (mut) Bcl-xLΔC, or 4 mg/ml Jurkat CE and added to mitochondria. After an additional incubation at 37°C for 1 h, mitochondria (pellet) and supernatant were probed for cytochrome c by Western blot.