FIG. 6.

crBAP31 inhibits Fas-mediated release of cytochrome c from mitochondria. (A) Control (parental) KB epithelial cells and KB cells stably expressing crBAP31-Flag were stimulated with anti-Fas (αFas/CHX) for the indicated times and examined by immunofluorescence confocal microscopy with mouse monoclonal antibody 2G8.B6 (anti-cytochrome c) and anti-mouse IgG coupled to Texas red. Cytochrome c release from mitochondria can be observed in cells prior to membrane blebbing (arrow indicates an obviously apoptotic cell). (B) At the indicated times of treatment, cells were homogenized and the postnuclear supernatant was separated into membranes (100,000 × g pellet [P]) and supernatant (S), as indicated, and equal aliquots were subjected to SDS-PAGE and immunoblotting with mouse monoclonal antibody 7H8.2C12 (anti-cytochrome c) (Cyt c). (C) Cells were treated with or without anti-Fas (α Fas) for 16 h, stained with DiOC₆, and subjected to FACS analysis. The arrow indicates the peak of fluorescence intensity obtained for cells in which the mitochondrial electrochemical potential was collapsed following treatment with 1 μM carbonyl cyanide m-chlorophenylhydrazone.