Fig. 6. Expression of the full-length HRSL3 protein (HREVFL) was controlled by incubation with the HRSL3-specific antibody (H-REV107-1). HRSL3 regulates PKC, which is required for HRSL3-induced apoptosis. (A) Phosphorylation of the indicated signaling proteins was analyzed by immunoblotting using antibodies specific for their phosphorylated forms. Immunoblotting against total amount of the proteins was used as a loading control. Expression of the HRSL3 protein, full-length and truncated form, was controlled by incubation with the HRSL3-specific antibody. (B) OVCAR-3 cells were either treated with 1, 10 and 20 nM of OA and with an inhibitor of the PI 3-kinase (LY294002; LY), or transfected with HRSL3. To determine the concentration of OA that inhibits PP2A activity only partially, phosphorylation of MEK1/2 and ERK1/2 kinases was examined using the corresponding antibodies (left panel). Then caspase-9 cleavage was analyzed (right panel). Actin- and caspase-9-specific antibodies were used as loading controls. (C) Phosphorylation of PKC is induced by treatment with both OA and HRSL3. Cells were treated with 10 nM OA, with DMSO or transfected with HRSL3 or the empty vector. Phospho-PKC or total PKC were measured by western blotting using the appropriate antibodies. (D) Twelve hours prior to transfection, OVCAR-3 cells were treated with the myristoylated PKC-specific pseudosubstrate. Then the cells were transfected with HRASL3 expression plasmid and caspase cleavage was analyzed 48 hours after transfection.