Figure 4. EPO promotes PRAS40 phosphorylation through PI 3-K and Akt during OGD exposure.

(A) Western blot analysis was performed for phosphorylated (p-)-PRAS40 (p-PRAS40, Thr²⁴⁶) in SH-SY5Y cells at 1, 3, or 24 hours (hrs) following OGD exposure. EPO (10 ng/ml) applied to cell cultures 1 hour prior to OGD maintained p-PRAS40 expression at significant levels over 24 hours following OGD (*P<0.01 vs. Control; ^† P<0.01 vs. OGD at corresponding time points). (B) EPO (10 ng/ml) applied to SH-SY5Y cells significantly increased the expression of phosphorylated (p-) p-PRAS40 3 hours later. EPO (10 ng/ml) applied to SH-SY5Y cultures 1 hour prior OGD significantly increased the expression of p-PRAS40 3 hours following OGD (*P<0.01 vs. untreated control; ^† P<0.01 vs. OGD treated alone). (C) EPO (10 ng/ml) was incubated with recombinant PRAS40 protein for 30, 60, and 180 min. No significant expression of phosphorylated (p-) PRAS40 was detected. (D) EPO (10 ng/ml) or EPO combined with the P I3-K inhibitors wortmannin (500 nM) or LY294002 (20 μM) were applied to SH-SY5Y cells and western blot analysis for phosphorylated (p-) p-PRAS40 and p-Akt1 (p-Akt1, Ser⁴⁷³) was performed 3 hours later. Wortmannin or LY294002 prevented the expression of p-PRAS40 and p-Akt1 during EPO (10 ng/ml) administration (*P<0.01 vs. untreated control; ^† P<0.01 vs. EPO treated alone). (E) EPO (10 ng/ml) was applied to SH-SY5Y cells 1 hour prior to OGD and western blot analysis for phosphorylated (p) p-PRAS40 and p-Akt1 was performed 3 hours following OGD. EPO significantly increased the expression of p-PRAS40 and p-Akt1 during OGD exposure. Wortmannin or LY294002 prevented the phosphorylation of PRAS40 and Akt1 during EPO administration following OGD (*P<0.01 vs. OGD treated alone; ^† P<0.01 vs. EPO/OGD). (F) Transfection of Akt1 siRNA prior to the application of EPO (10 ng/ml) in SH-SY5Y cells significantly limited the expression of Akt1 and significantly reduced the expression of phosphorylated (p-) p-PRAS40 3 hours after administration of EPO. Scrambled siRNA transfection did not alter the expression of Akt1 and p-PRAS40 during EPO application (*P<0.01 vs. untreated control; ^† P<0.01 vs. EPO treated alone). (G) Akt1 siRNA was transfected into SH-SY5Y cells prior to EPO (10 ng/ml) application and OGD exposure. Western analysis expression of phosphorylated (p-) p-PRAS40 and Akt1 was determined 3 hour following OGD. EPO (10 ng/ml) increased p-PRAS40 expression following OGD. Transfection of Akt1 siRNA significantly limited p-PRAS40 expression during OGD alone and during EPO treatment with OGD (*P<0.01 vs. OGD; ^† P<0.01 vs. EPO/OGD). In all cases above, each data point represents the mean and SD from 3 experiments.