Fig. 5.

The inhibition of mTORC1 is PKA dependent. A) TSC2^−/− MEFs and B) HEK293 cells were serum-starved in DMEM for 16 h. For A), cells were incubated in KRB supplemented with 0.5× amino acids. For B), cells were incubated in KRB supplemented with 20 mM glucose but in the absence of amino acids. For both A) and B), after 30 min, cells were treated with increasing concentrations of H89, a PKA inhibitor, for 30 min, followed by treatment with forskolin (10 μM) and IBMX (1 mM) for 30 min, before stimulation with amino acids (5×) for a further period of 30 min (for B)). Proteins were resolved on SDS-PAGE and Western blotted using antibodies against phosphorylated (P)-S6K1 Thr389 (T389), P-rpS6 Ser235/236 (S235/236), and as a loading control, rpS6. All results are representative of three independent experiments.