Figure 6.

Par-4 is highly phosphorylated on S231 (ortholog of rodent S223) in human prostate cancer cells compared with normal counterparts. (a) Upper panel, phosphorylation of Par-4 on S231 was studied by western blotting in prostate cancer cells (PC-3, LnCap cells) and in normal prostate cells (PrCE, PNT2C2). In parallel, endogenous expression of Par-4 and CK2 subunits in the different cell lines was determined (middle panel). Hsp90 was used as a loading control. Lower panel, analysis of CK2 activity in the different prostate cells studied using Cyclex CK2 screening kit. Bars represent the mean±S.D. of at least two independent experiments. (b) PC-3 cells were transfected with CK2 siRNA (siCK2T, ThermoFischer) or scrambled siRNA fluorescently labeled with FAM (Scr siRNA) for 48 h and then treated or not with TRAIL (500 ng/ml, 3 h). Endogenous phosphorylated human Par-4 was detected by western blotting using the anti-phospho231 Par-4 antibody (Ph231-hPar-4) (left panel). Par-4 expression (left panel) and downregulation of CK2α protein (right panel) were evaluated by immunoblotting using the corresponding antibodies. Bar graph shows semi-quantified densitometry from western blotting analysis