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. 2012 Sep 18;7(9):e45456. doi: 10.1371/journal.pone.0045456

Figure 2. EPO phosphorylation of mTOR, p70S6K, and 4EBP1 is dependent upon PI 3-K and Akt.

Figure 2

(A) Rapamycin (50 nM) administration or mTOR siRNA transfection during OGD exposure alone or treatment with EPO (10 ng/ml) applied 1 hour prior to OGD prevented the phosphorylation (p-) of p-mTOR, p-p70S6K, and p-4EBP1 3 hours following OGD exposure. Transfection with mTOR siRNA also significantly limited the expression of total mTOR (*P<0.01 vs. OGD treated alone; † P<0.01 vs. EPO/OGD). (B) EPO (10 ng/ml) was incubated with recombinant mTOR protein (1 μg) for 30, 60, and 180 min. No significant expression of phosphorylated (p-) mTOR was detected. (C) Akt1 siRNA transfection in SH-SY5Y cells prior to OGD or prior to treatment with EPO (10 ng/ml) applied 1 hour prior to OGD significantly reduced the expression of total Akt1, phosphorylated (p-) p-mTOR, p-p70S6K, and p-4EBP1 3 hours following OGD (*P<0.01 vs. OGD treated alone; † P<0.01 vs. EPO/OGD). (D) EPO (10 ng/ml) or EPO combined with wortmannin (500 nM) or LY294002 (20 μM) was applied to SH-SY5Y cells. Western blot analysis was performed to detect the expression of phosphorylated (p-) p-p70S6K and p-4EBP1 3 hours later. EPO phosphorylation (p-) of p-p70S6K and p-4EBP1 was blocked by the PI 3-K inhibitors wortmannin or LY294002 (*P<0.01 vs. EPO treated alone). (E) 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 western blot was performed to detect the expression phosphorylated (p-) p-p70S6K and p-4EBP1 3 hours following OGD. EPO phosphorylation (p-) of p-p70S6K and p-4EBP1 during OGD was blocked by the PI 3-K inhibitors wortmannin or LY294002 (*P<0.01 vs. OGD treated alone; † P<0.01 vs. EPO/OGD). In all cases above, each data point represents the mean and SD from 3 experiments.