FIGURE 5.

ERK1/2 phosphorylation mediates mTOR activation in a tuberin-independent manner. A and B, phosphorylation of mTOR and p70S6K was checked by Western blot analysis after TGF-β1 treatment for 6 h. Pretreatment with U0126 and PD184352 reduced mTOR and p70S6K activation (n = 3). C, densitometry analysis of B. D, Western blot analysis result showing that treatment of TGF-β1 for 6 h increased phosphorylation of TSC2 (Ser⁶⁶⁴), which was blocked by U0126. The total cell lysate from HeLa cell treated with nocodazole (10 μm) for 1 h was loaded as a positive control (n = 4). E, Western blot analysis for p-Akt (Ser³⁰⁸), p-TSC2 (Ser⁹³⁹ and Thr¹⁴⁶²), and t-TSC2. Wortmannin (50 and 100 nm) inhibited TGF-β1-induced Akt and TSC2 phosphorylation. F, Western blot analysis showing that 100 nm wortmannin blocked insulin-induced p70S6K phosphorylation but not TGF-β1-induced p70S6K phosphorylation (n = 3). G, Western blot analysis showing that siTSC2 did not reverse TGF-β1- and insulin-induced p70S6K phosphorylation and that addition of U0126 and rapamycin completely prevented TGF-β1-induced p-p70S6K in siTSC2-treated groups (n = 3). Values are mean ± S.E. *, p < 0.05.