FIGURE 7.

Inhibitory effects of Z-YVAD-FMK and Z-IETD-FMK on Cholix-induced apoptotic signals. Cells were grown overnight and then pretreated with the indicated inhibitors (50 μm). After 40 min, cells were incubated for 18 h with wild-type (WT) or mutant (MT) Cholix (10 μg/ml) in the presence or absence of the inhibitor. Total cell lysate (TCL) was collected from cells and resolved by immunoblotting to detect the indicated caspases cleavages or PARP cleavage. The cytosolic fraction was collected as described in the previous report (18). Cytochrome c release into cytosol was detected using an anti-cytochrome c antibody. Mean values were calculated on relative band intensity in three separate experiments. The utilized inhibitors were represented as follows; YVAD, caspase-1, -4, and -5 inhibitor Z-YVAD-FMK; and IETD, caspase-8 inhibitor Z-IETD-FMK.