Fig. 5. Rat mitochondrial Bak is digested by trypsin after tBid treatment, but mitochondrial cytochrome c is resistant. (A) Mitochondria were incubated with or without 5 nM tBid for 30 minutes and then with the indicated concentration of trypsin at 0°C for 10 minutes. Western blots were probed with anti-Bak antibody that only recognises uncleaved Bak. (B) The Bak signal was used to calculate the level of undigested Bak. (C) Mitochondria were incubated with or without 5 nM tBid for 9 minutes (yielding partial release of cytochrome c) and then with trypsin, as indicated, for 10 minutes at 0°C. Mitochondria were sedimented and their cytochrome c analysed in immunoblots.