BAX α9 mutants show reduced activity when expressed in cells. (A) BAX structure (PDB: 1F16) showing the positions of the BAX missense mutations tested in this study (red). BAX helix α9 is highlighted in blue. (B) Test of activity of BAX mutants K123M, A183P, A183T, S184*, W188* in cells. BAX/BAK DKO MOLM-13 cells expressing the indicated BAX variants were treated with increasing concentration of venetoclax (50-5000 nM) for 24 hours, and cell viability was determined by Annexin V/DAPI staining and flow cytometry. Data are means ± SEM of 3 independent experiments. (C) Upon venetoclax treatment, BAX α9, but not BAX K123M, show reduced exposure for conformation-specific epitope 6A7. MOLM-13 DKO cells expressing the different BAX variants were pre-incubated with 25 μM Q-VD oph for 1 hour and then treated with 500 nM venetoclax. After 5 hours, cells were permeabilized with 0.025% digitonin prior to staining with conformation-specific anti-BAX antibody 6A7. After several washes, samples were stained with a PE-conjugated secondary antibody and analyzed by flow cytometry. The results are representative of at least two independent experiments. (D) Mutations in BAX α9 reduce MOM translocation and integration of BAX. Cells were treated as described in (C), subjected to carbonate extraction and fractions run on SDS-PAGE and immunoblotted for BAX. The results are representative of three independent experiments.