Figure 6

Lysine 21 and 64 of Bax are required for parkin regulation of Bax translocation into the mitochondria. (a) Whole-cell lysates of CHO cells transiently expressing wild-type human Bax (hBax) or each of its mutants and probed with the human-specific 2D2 antibody by Western blot. (b) Representative images of cells co-transfected with mito-mCherry (mitochondrial marker) and GFP-Bax, showing the various stages of Bax translocation into the mitochondria after treatment with 1 μM staurosporine for 5 h. Scale bar, 10 μm. (c) CHO cells without or with stable expression of parkin (CHO-Parkin) were transiently transfected with wild-type GFP-Bax or each of its GFP-tagged mutants. Representative images of cells showing Bax translocation into the mitochondria after treatment with DMSO or 1 μM staurosporine (Sts) for 5 h. Scale bar, 20 μm. (d) Quantification of b. Cells were scored visually for Bax translocation into the mitochondria as completely cytoplasmic (open), predominantly cytoplasmic (open dotted), predominantly mitochondrial (gray) or completely mitochondrial (gray dotted) by a blinded observer. Hundred and five to 160 cells were counted per condition. Data were plotted from three independent experiments. (e) Percentage of pooled mitochondrial fractions (predominantly mitochondrial+completely mitochondrial) plotted from d. All bars represent mean±S.D., n=3. Asterisk indicates significant differences when compared with WT, # indicates significant difference between K21R and K21R/K64R, † indicates significant difference between K64R and K21R/K64R (P<0.05)