Figure 3

Cleavage of Par-4 by caspase-3 in vitro . Panel A , Effect of pan caspase inhibitor on the cleavage of Par-4. Jurkat cells were pre-treated with z-VAD-fmk at the indicated concentration for 1 h. The cells were then treated with SPH (8 μM) for 6 h and the lysate were immunoblotted with Par-4 and PARP antibodies. Actin was used as the loading control. Panel B , Jurkat cells were pre-treated with z-VAD-fmk at the indicated concentration for 1 h, then the cells were treated with SPH (8 μM) for 6 h and DNA fragmentation was analysed. Panel C , Jurkat cells were pre-treated with z-VAD-fmk at the indicated concentration for 1 h, then the cells were treated with SPH (8 μM) for 6 h and cell viability was measured by using WST viability assay kit. Data shown are means ± SD (n = 3). *, p < 0.05, and **, p < 0.05 compared with untreated control and SPH treated cells, respectively. Panel D , Immuno purified Par-4 was incubated with 300 ng of each purified caspase-3, -7, and −8 for 2 h. The reaction was stopped by adding SDS-PAGE sample buffer and proteolytic cleavage of Par-4 was detected by using anti-Par-4 antibody. Panel E , Immuno purified Par-4 was incubated with 300 ng of purified caspase-3 in the presence and absence of caspase-3 specific inhibitor (Ac-DEVD-CHO). The reaction was stopped by adding SDS-PAGE sample buffer and proteolytic cleavage product of Par-4 was detected by using anti-Par-4 antibody.