(A) Mitochondrial fractions from cells expressing the indicated cysteine variants were incubated with tBid (or heated to 43°C) to oligomerise Bak, or Bax, and oxidant (CuPhe) added to induce disulphide bonds where indicated. Aliquots were analyzed by BNP, non-reducing SDS PAGE, and immunoblotted for Bak or Bax. Variants lacking cysteines (Cys null) were included to show the altered migration of Bak and Bax complexes on BNP in the absence of disulphide bond formation. Data are representative of at least two biological replicates. Note that in the left panels, Bak V61C exhibits the same CuPhe linkage pattern with both tBid (lane 4) and heat treatment (lane 5). In the right panels, linkage between A46C in a mitochondrial form of Bax efficiently links between dimers. Bax S184L/A46C is a Bax variant that is constitutively membrane localised (due to the hydrophobic substitution of S184L in the transmembrane domain [Nechushtan et al., 1999; Fletcher et al., 2008]) with a N-terminal cysteine substitution (A46C) analogous to Bak V61C. Bax exhibits laddering on BNP in the absence of CuPhe treatment, but addition of CuPhe induces efficient linkage between Bax dimers at residue A46C, resulting in the disappearance of dimers and appearance of high molecular weight complexes on BNP (lane 9). Thus, we predict Bax dimers exhibit similar flexible extremity characteristics as Bak dimers at the MOM. (B) Pellet and supernatant fractions were further analysed for cytochrome c release, and confirmed each variant was responsive to tBid (and heat treatment in the case of Bak V61C). Data are representative of at least two biological replicates.