Effect of AICAR on insulin-stimulated phosphorylation of the PI3K pathway. 3T3-F442a preadipocytes were differentiated into adipocytes. A. The cells were treated with or without AICAR (AI) (1 mM, 2 h), followed by insulin (Ins) (100 nM, 15 min) and equal amounts of cell lysates were immunoprecipitated with antibodies against IRS1 and assayed on the associated PI3K activity. The radiolabeled PIP3 lipids were resolved on thin layer chromatography and exposed to X-ray film. The autoradiogram represents one of a triplicate experiment. The PIP3 spots were excised and counted in liquid scintillation. The graph denotes the average CPM of labeled PIP3 (n = 3, mean ± SD). B. In parallel to A, protein samples were run onto SDS-PAGE and blotted with antibodies, as indicated.