Figure 6.
Endogenous Bax accumulates on the mitochondria in the absence of retrotranslocation. (a) Western blot analysis of endogenous Bax localization in Mcl-1 knockout (KO) mouse embryonic fibroblasts (MEFs) after 6 h treatment with 0, 0.01, 0.1 and 1 μM ABT-737 (from left to right, respectively). Cytosol (C) and heavy membrane fraction (HM) of Mcl-1 KO cells are displayed. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and Tom20 serve as controls for the fractionation. (b) Quantification of endogenous Bax levels in the C and in the HM is dependent on the application of different ABT-737 concentrations revealed by western blot. P-values according to a one-way analysis of variance (ANOVA) test are depicted (n=7). (c) Analysis of endogenous Bax localization in HCT116 wt cells either overexpressing different Bcl-xL variants or after 6 h treatment with 1 μM ABT-737 by western blot. Apoptosis induction by 1 μM staurosporine (STS) serves as control. C and HM of HCT116 wt cells are displayed. GAPDH and Tom20 serve as control for equal protein loading. (d) Quantification of endogenous Bax levels in the C and in the HM of HCT116 cells either overexpressing different Bcl-xL variants or after treatment with 1 μM ABT-737 or 1 μM STS as analyzed by western blot (n=3). (e) Western blot analysis of carbonate extraction of membrane-associated endogenous Bax in HCT116 wt cells either overexpressing different Bcl-xL variants or after administration of 1 μM ABT-737 or 1 μM STS. Displayed are supernatant (S) and pellet (P) of the carbonate extraction. VDAC2, Tom20 and Smac served as controls