EPO promotes the binding of PRAS40 to protein 14-3-3 through PI 3-K.

<p>(<b>A</b>) EPO (10 ng/ml) or EPO combined with wortmannin (500 nM), LY294002 (20 μM) or rapamycin (50 nM) was applied to SH-SY5Y cells and cell extracts 3 hours later were immunoprecipitated by antibodies against protein 14-3-3 or mTOR. Western blot analysis was performed to detect the expression phosphorylated (p-) p-PRAS40 and total PRAS40 in the precipitates (*<i>P</i><0.01 vs. untreated control; ^†<i>P</i><0.01 vs. EPO treated alone). Application of EPO increased the expression of p-PRAS40 in the precipitate. In contrast, wortmannin or LY294002 significantly reduced expression of p-PRAS40 in the precipitate. (<b>B</b>) EPO (10 ng/ml) or EPO combined with wortmannin (500 nM) or LY294002 (20 μM) was applied to SH-SY5Y cells 1 hour prior to OGD and cell extracts 3 hours following OGD were immunoprecipitated by antibodies against protein 14-3-3 or mTOR. Western blot analysis was performed to detect the expression phosphorylated (p-) p-PRAS40 and total PRAS40 in the precipitates (*<i>P</i><0.01 vs. OGD; ^†<i>P</i><0.01 vs. EPO/OGD). Application of EPO increased the expression of p-PRAS40 in the precipitate during OGD. In contrast, wortmannin or LY294002 significantly reduced expression of p-PRAS40 in the precipitate during OGD. In all cases above, each data point represents the mean and SD from 3 experiments.