Fig. 2. Disassembly of OPA1 complex by BH3-only proteins required the presence of Bak or Bax.

a. N/C-Bid-induced cytochrome c release and OPA1 complex disassembly, in Bak-deficient mouse mitochondria. Isolated liver mitochondria from Bak-null mice were treated with either 10 nM N/C-Bid, 100 nM Bcl-x_L or both or left untreated for 30 min at 37° C. Samples were centrifuged, and pellets were treated for 45 min in 10 mM EDC in PBS at 37° C before the addition of SDS buffer. Proteins in the pellets and supernatants were separated on 4–12% polyacrylamide gradient gels and analyzed by immunoblotting using anti-OPA1, -HAX1 and -cytochrome c antibodies. b. Recombinant oligomerized Bax induced disassembly of Opa1 complexes in Bak-deficient mitochondria. Mitochondria from Bak-null mice were incubated either with 10 nM tBid, 10 nM oligomerized Bax, 100 nM BaxΔC or left untreated, with or without 100 nM Bcl-x_LΔC for 30 min. Bcl-x_LΔC at this concentration is not expected to block activated Bax. c and d. Opa1 disassembly induced by Bid required Bak or Bax. Mitochondria from WT (c) or from Bax/Bak double-knockout (d) MEFs were treated with N/C-Bid, Bcl-x_LΔC or both or left untreated for 30 min. at 37° C.