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Figure 4 | Journal of Molecular Signaling

Figure 4

From: Caspase-3 mediated release of SAC domain containing fragment from Par-4 is necessary for the sphingosine-induced apoptosis in Jurkat cells

Figure 4

Identification of the cleavage site. Panel A , D126A, D131A, D175A, D179A, D180A, and D191A mutant were constructed. Each mutants were transiently transfected into HEK-293 cells. Cells were harvested 1 day after transfection and the cytosolic fraction of each transfectants were incubated with caspase-3 for 2 h in vitro. The proteolytic cleavage product of Par-4 was detected by anti-DDK monoclonal antibody. The activity of caspase-3 was also confirmed by checking the PARP cleavage. Actin was used as the loading control. Panel B , Wild type and each mutant Par-4 was transiently transfected in to HEK-293 cells and the cells were treated with either vehicle or 50 μM Cur for 24 h. The proteolytic cleavage of each mutant Par-4 was detected by immuno-probing of each lysate with anti-DDK monoclonal antibody. Apoptosis induction was confirmed by PARP cleavage and actin was used as protein loading control. Panel C , Schematic representation of the release of SAC domain containing fragment from the Par-4 during the apoptosis. Nuclear localization sequences (NLS1 and NLS2), selective for apoptosis induction in cancer cells (SAC). Panel D , Alignments of the regions containing the caspase-3 cleavage site of Par-4 from different species.

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