<p>(<b>A</b>) Cytosolic and mitochondrial Bak/BaxCS both contribute to permeabilization of MEF mitochondria. Cytosol and membrane fractions from <i>bak<sup>−/−</sup>bax<sup>−/−</sup></i> MEFs or from <i>bak<sup>−/−</sup>bax<sup>−/−</sup></i> MEFs expressing either Bak or Bak/BaxCS were combined as shown, and incubated at 30°C in the presence of 100 nM tBid for 0, 30 or 60 mins, or without tBid for 60 mins (60-). Supernatant (<i>Cyt</i>) and membrane (<i>Memb</i>) fractions were immunoblotted for cytochrome <i>c</i> and for Bak. (<b>B</b>) Cytosolic Bak/BaxCS is sufficient to permeabilize mouse liver mitochondria. MEF cytosol fractions derived as in (A) were combined with mitochondria isolated from wild-type (<i>bak<sup>+/+</sup></i>) or <i>bak<sup>−/−</sup></i> mouse liver, and incubated at 37°C with or without 100 nM tBid for 60 min. Supernatant (<i>Cyt</i>) and membrane (<i>Memb</i>) fractions were immunoblotted for cytochrome <i>c</i> and for Bak. Note that Bak/BaxCS levels appear high compared to the endogenous mouse Bak, possibly due to different recognition by the anti-Bak antibody. (<b>C</b>) Mcl-1 is higher in MEF membranes than in mouse liver mitochondria. MEF membranes and mouse liver mitochondria at the concentrations used in (A) and (B) were examined for levels of Mcl-1, Bcl-x<sub>L</sub> and VDAC1 by immunoblotting. Results are representative of two or more independent experiments.</p
