Figure 4. Bak dimer-dimer interactions are disrupted by detergent or Bax.
(A) Digitonin prevents linkage between Bak dimers. Membrane fractions expressing the indicated Bak single-cysteine variants were first treated with tBid, and then supplemented, as indicated, with detergent (1% digitonin) prior to cysteine linkage. Two additional mutants were included to show linkage within dimers at the BH3:groove interface (M71C/K113C) and at extensions to the C-terminus (GGSGGCK). Data are representative of two biological replicates. (B) Bax can disrupt Bak dimer-dimer association. Membrane fractions first treated with tBidM97A were then incubated, as indicated, with Bax, Bcl-xL, Mcl-1ΔN151ΔC23 or digitonin prior to cysteine linkage (upper panels). Supernatant and pellet fractions showed partial localisation of each recombinant protein to the membrane fraction (lower panels). Data are representative of three biological replicates.