Fig. 7: Dependence of BMF- and HRK-induced apoptosis on the alternative BAK binding mode.

a Liposome permeabilization assay performed in the presence of 50 nM wild-type (WT) BAKΔTM or BAKΔTM F161A and 50 nM of the indicated BH3 peptides. p = 0.24, 0.33, 0.20, 0.006, and 0.003 for control, BIM, PUMA, BMF, and HRK BH3s, respectively, when liposome release mediated by BAK F161A mutant was compared with WT BAK in the presence of the same BH3 peptide. b Bak^−/−Bax^−/− MEFs were transduced with the indicated retrovirus to express WT BAK and BAK F161A. After 2 weeks of selection, a pool of cells was subjected to western blot. c–e After the indicated EGFP-tagged BH3-only proteins were transfected into Bax^−/−Bak^−/− DKO MEFs reconstituted with WT BAK, F161A BAK c, e or empty vector (E.V.), BAK G126S without or with the reciprocal N86G mutation that restores BAK oligomerization³⁸ d, cells were harvested, stained with APC-conjugated Annexin V, and subjected to flow cytometry c, d. The percentages of EGFP⁺ cells that are Annexin V⁺ are indicated. p = 0.18, 0.0006, 0.0002 for PUMA, BMF, and HRK, respectively, when the percentages of Annexin V⁺ cells reconstituted with BAK F161A were compared with those reconstituted with WT BAK c, and p = 0.10, 0.0009, 0.001 for BIM, BMF, and HRK, respectively, when the percentages of Annexin V⁺ cells of reconstituted with BAK G126S/N86G were compared with those reconstituted with WT BAK d. In addition, cell lysates were prepared for immunoprecipitation with BAK Ab-1 antibody and blotting for BAK e. f After S-tagged WT or F161A BAK was transfected into MEFs for 24 h, cell lysates were subjected to pull-down with S-protein agarose and blotting with the indicated antibodies. g Anoikis was induced for 48 h in Bak^−/−Bax^−/− DKO MEFs reconstituted with WT BAK or BAK F161A. The percentage of cells with sub-G1 DNA was assayed (p = 0.0006). Right panels in c and d, whole-cell lysates subjected to immunoblotting. Error bars: mean ± S.D. of three independent experiments. ns, p > 0.05; **p < 0.01; ***p < 0.001, paired two-tailed t test for comparison of dextran release or cell death induced by mutants vs WT. Source data are provided as a source data file.