Fig. 6. GSK-3 activation is mediated through PP2A-regulated inactivation of the PI 3-kinase-Akt pathway. (A) 10I cells were treated with 25 µM of C₂-ceramide for 4 hours with or without LiCl, wortmannin (10 nM) or PD98059 (100 µM). We used western blotting to determine GSK-3 phosphorylation (P-GSK-3) at serine 9; the relative ratio of P-GSK-3: total GSK-3 protein level is shown. -actin expression was an internal control. (B) 10I cells were transfected with 50 nM (88.7% transfection efficiency) of Akt siRNA as described in Materials and Methods. After ceramide treatment, levels of P-GSK-3, GSK-3 and Akt were detected using western blotting. The relative ratio of P-GSK-3:GSK-3 protein level is shown. -actin levels were determined as an internal control. (C) 10I cells were treated with 25 µM of C₂-ceramide with or without the PP2A inhibitor OA for different time periods as indicated. We used western blotting to determine phosphorylated GSK-3 (P-GSK-3) and Akt phosphorylated at serine 473 (P-Akt); the relative ratios of P-Akt:total Akt and P-GSK-3:GSK-3 are shown, respectively. -actin expression was an internal control. (D) To determine whether PP2A has a direct effect on Akt dephosphorylation, Akt was immunoprecipitated (IP) from untreated cells and then incubated together with PP2A that had been immunoprecipitated from 25 µM of ceramide-treated cells for 30 minutes at 30°C with or without 50 nM of OA. After washing, we used western blotting to determine P-Akt; the relative ratio of P-Akt:IP Akt protein levels is shown.