FIGURE 6:

Validation of RSK1/2-dependent TSC2 phosphorylation at serine 1798. (A) 293T cells were treated with increasing concentrations of rapamycin, Torin 1, or the AKT1 inhibitor MK2206 for 1 h. Whole-cell extracts were immunoblotted as indicated. (B) 293T cells were serum starved overnight and were then treated with rapamycin, Torin 1, or MK2206 as indicated for 1 h prior to PMA addition for 15 min. After the 15-min PMA treatment, cells were harvested on ice, and whole-cell extracts were immunoblotted as indicated. Filled black circles indicate the following drug concentrations: PMA, 1 μM; rapamycin, 100 nM; torin-1, 100 nM; MK2206, 1 μM. Black outlined gray circles indicate the following drug concentrations: PMA, 1 μM; rapamycin, 250 nM; torin-1, 250 nM, MK2206, 5 μM. (C) 293T cells were serum starved overnight and were then treated with Torin-1 or RSK inhibitor (RSKi, BI-D1870) as indicated for 1 h prior to PMA addition for 15 min. Whole-cell extracts were immunoblotted as indicated. Filled black circles indicate the following drug concentrations: PMA, 1 μM; torin-1, 100 nM; RSKi, 10 μM.