Fig. 1.

cAMP inhibits insulin signalling to mTOR. A) and B) HEK293 cells or C) and D) TSC2^+/+ MEFs were serum starved in DMEM for 16 h before preincubated in KRB for 30 min, then incubated in KRB with or without forskolin (Fsk) (10 μM in A/B) plus IBMX (1 mM) for 30 min, before treatment with insulin (100 nM) or TPA (1 μM) for a further 30 min as indicated. A) and C) Proteins were resolved by SDS-PAGE and Western blotted using antisera against phosphorylated (P)- rpS6 Ser240/Ser244 (S240/244), Ser235/Ser236 (S235/236), 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. S.E.: short exposure; L.E.: long exposure. [cAMP]_i levels in B) and D) were determined and expressed relative to cellular protein content, and shown as means ± SE; n = 3. In B), ***P < 0.001 versus time-matched control (no Fsk and IBMX) as indicated. There was no significant effect of insulin treatment on cAMP levels. For clarity, not all differences are shown. In C), ***P < 0.001 as indicated. Statistical analyses were by Bonferroni's test following one-way ANOVA. Immunoblots are representative of three independent experiments.