Fig. 3.

AMPK is not involved in the inhibition of mTORC1 by cAMP. A) TSC2^+/+ and B) TSC2^−/− MEFs were infected with the recombinant adenovirus expressing dominant negative (DN) AMPK for 32 h. Cells were serum starved in DMEM for 16 h before preincubation in KRB for 30 min, and then treated with or without forskolin (10 μM) plus IBMX (1 mM) for 30 min, followed by a further 30 min incubation with insulin (100 nM) or AICAR (1 mM) as indicated. Cell lysates were separated by SDS-PAGE and subjected to immunoblotting with antisera against phosphorylated (P)- S6K1 Thr389 (T389), P-AMPK Thr172 (T172), P-4EBP1 Ser65 (S65), as well as AMPK, rpS6 and 4EBP1. All results are representative of three independent experiments.