Loss of RASSF2 impairs TRAIL-induced PAR-4 nuclear trafficking in prostate cancer cells. PC-3 cells stably expressing a RASSF2 shRNA construct or control vector were transfected with GFP-PAR-4 and treated with 100 ng/ml TRAIL for 1 h. (A) Cells were lysed, and nuclear (Nuc) and cytoplasmic (Cyt) fractions were prepared and analyzed by Western blotting for GFP-PAR-4. Densitometric quantitation of Western blot results is shown. Bars show means of triplicate experiments, with standard deviations indicated. *, statistically different (P < 0.01) from untreated cells. (B) Representative Western blot. TFIIH and p38 were used as markers for the nuclear and cytoplasmic fractions, respectively. Similar experiments were performed for the endogenous PAR-4 protein. (C) Densitometric quantitation of the Western blot results with endogenous PAR-4 in nuclear and cytoplasmic fractions in the RASSF2 (positive and negative) PC-3 cells. Bars show means of triplicate experiments, and standard deviations are indicated. *, P < 0.01 compared to untreated cells.