FIGURE 3

gzmB⁺CTL-induced conformational change of Bak and Bax depends on Bid and is critical for cytochrome c release independent of caspases. Gp33-pulsed MEF.wt (see Materials and Methods), MEF.Bid^-/- (a, c) and MEF.Bak Bax^-/- (a) or MEF.wt (see Materials and Methods), MEF.Casp 3 7^-/- and MEF.Casp 9^-/- (b) cells were incubated with ex vivo virus-immune gzmB⁺CTL (4 h, 10:1 effector/target ratio). Cytochrome c release from mitochondria (a, b) and Bak and Bax conformational change (c) were analyzed by three-color flow cytometry in the cell population negative for CD8 expression (target cells) as described in Materials and Methods. Histograms show a representative experiment of two independent experiments. Ctr: only target cells. (d) Gp33-pulsed MEF.wt cells were incubated with ex vivo virus-immune B6 or gzmB^-/-CTL (1 h, 10:1 effector/target ratio). Subsequently, lysates were prepared and Bid cleavage was analyzed by western blot as described in Materials and Methods. Bid=22 kDa, tBid=16 kDa