Figure 5.

L70A/D71A, but not other Bax mutants, loses the ability to multimerize, interact with each other, and induce mitochondrial membrane potential change and caspase activation. (a) HCT116 cells with indicated BAX genotypes were treated with 10 ng/ml TRAIL for 24 h. Mitochondrial membrane potential was analyzed by staining cells with MitoTrcker Red CMXRos, followed by flow cytometry. (b) Cells with indicated BAX genotypes were treated with TRAIL as in (a). Activation of caspase 9 was analyzed by western blotting. Arrowheads indicate active caspase 9. (c) Mitochondrial fractions were isolated from cells with different BAX genotypes treated with 120 μℳ sulindac for 36 h. Bax multimerization was analyzed by treating mitochondrial fractions with the chemical cross-linker DSP, followed by western blotting under non-denaturing conditions. Cox IV and β-actin were used as control for loading and fractionation. (d) BAX-KO cells were cotransfected with HA-tagged and GFP-tagged WT Bax or HA-tagged and GFP-tagged L70A/D71A mutant. IP was performed to analyze the interaction of Bax with each other