Fig. 5. Ceramide activates PP2A, which then indirectly activates GSK-3. (A) 10I cells were treated with 25 µM C₂-ceramide for 6 hours with or without LiCl or OA. We used western blotting to determine phosphorylated GSK-3 (P-GSK-3). Total GSK-3 protein was the control. The relative ratios of P-GSK-3:total GSK-3 protein are shown. -actin levels were determined as an internal control. (B) 10I cells were transfected with PP2A (0.02 and 0.1 µg) for 6 hours and incubated without or with OA for an additional 6 hours. The transfection efficiency determined using FITC-labeled immunoglobulin was 92.7% for the cells transfected with 0.02 µg PP2A and 95.8% for those transfected with 0.1 µg PP2A. We used western blotting to determine phosphorylated GSK-3; the ratio of P-GSK-3:total GSK-3 protein level is shown. -actin levels were determined as an internal control. (C) We used immunostaining plus confocal microscopy to determine phosphorylated GSK-3 (green), and PI staining (red) for nuclear staining. (D) To determine whether PP2A directly affects GSK-3 dephosphorylation, GSK-3 was immunoprecipitated (IP) from untreated cells and then incubated together with PP2A that had been immunoprecipitated from 25 µM ceramide-treated cells for 30 minutes at 30°C with or without 50 nM OA. After the cells had been washed, we used western blotting to determine P-GSK-3; the relative P-GSK-3:IP GSK-3 protein level is shown. The IP PP2A level was determined using western blotting. PP2A activity was determined using substrate dephosphorylation; relative PP2A activity is shown.