Fig. 2.

cAMP inhibits the activation of mTORC1 independently of TSC2. TSC2^−/− or TSC2^+/+ MEFs were serum starved in DMEM for 16 h before preincubation in KRB for 30 min, followed by incubation in KRB with or without forskolin (0.1–10 μM) plus IBMX (1 mM) for 30 min. Cells were then treated with insulin (100 nM) or TPA (1 μM) for a further 30 min as indicated. Cell lysates were separated by SDS-PAGE and subjected to immunoblotting with antisera against phosphorylated (P)- rpS6 Ser240/Ser244 (S240/244), P-S6K1 Thr389 (T389), P-4EBP1 Ser65 (S65), P-PKB Ser473 (S473), P-PRAS40 Thr246 (T246), P-Erk1/2, as well as S6K1, rpS6 and 4EBP1. Levels of P-S6K1 Thr389 and P-4EBP1 Ser65 were quantified by densitometric analysis and are presented as arbitrary units (A.U.). Results shown are means ± SE; n = 3. *P < 0.05, **P < 0.01, ***P < 0.001 by Dunnett's test following one-way ANOVA. Immunoblots are representative of three independent experiments.