Figure 3.

Phosphorylation of rodent Par-4 by CK2 blocks Par-4 proapoptotic functions. (a–c) PC-3 cells were transfected with empty GFP vector control (Mock), rat GFP-tagged Par-4, GFP-tagged 124A223A mutant (mimicking non-phosphorylated Par-4 form) or 124D223D mutant (mimicking phosphorylated Par-4 form) and then treated with recombinant death ligand TRAIL (500 ng/ml, 3 h). (a) Green fluorescent (transfected) cells were gated (represent around 20% of total cell) and further analyzed for apoptosis by Hoechst staining. Bars represent the mean±S.D. of at least four independent experiments. (b) FACS analysis of the caspase-3 activity was assessed by red fluorescent signal FLICA (Fluorogenic inhibitors of caspase-3 activation). Bars represent the mean±S.D. of at least four independent experiments. (c) Immunoblot analysis of caspase-8 and PARP cleavage (included both GFP-positive and -negative cells) at the indicated times. *P<0.05, **P<0.01. (d) PC-3 cells, transfected as above, were treated 8 h with 200 nM of Paclitaxel, and then apoptosis was determined by detection of caspases 8 and PARP cleavage. Hsp90 and 14-3-3 were used as a loading control