IL-10 activation of the PI3K/Akt pathway requires AMPK. (A and B), IL-10 activates PI3K and Akt in AMPKα1+/+ BMDM. BMDM generated from AMPKα1+/+ and AMPKα1−/− mice were treated with rm-IL-10 (20 ng/ml) for the time points indicated. Total cellular lysates were collected for Western blot assessment of (A) AMPK-Thr172, ACC-Ser79, PI3K-Tyr199, and (B) Akt-Thr308 and Akt-Ser473 phosphorylation. (C), PI3K inhibition does not block IL-10 activation of AMPK. BMDM generated from C57BL/6 mice were incubated with either media alone, or with LY294002 (20 µM) for 1 h, then exposed to rm-IL-10 (20 ng/ml) for the time points indicated. Total cellular lysates were collected for Western blot assessment of Akt-Thr308 and Akt-Ser473 and AMPK-Thr172 phosphorylation. Protein phosphorylation levels were analyzed by densitometry and are displayed as a bar histogram. Results shown are representative of three independent experiments.