Solenopsin inhibits insulin-stimulated PI3K signaling in 3T3-L1 cells. 3T3-L1 fibroblasts were treated with 30 μM solenopsin A for 20 minutes prior to stimulating with 1 μM insulin for 10 minutes. (A) Insulin-stimulated, IRS-1–associated PI3K activity was assessed by measuring the incorporation of labeled phosphate from [γ³² P]ATP into phosphatidylinositol. Labeled phosphatidylinositol 3-phosphate was resolved from ATP by thin-layer chromatography, which was visualized using a Storm Phosphorimager. Intensity of the bands was quantified by ImageQuant software (Molecular Dynamics, Sunnyvale, CA). (B) Cell lysates were resolved by SDS-PAGE, Western-blotted with anti–phospho-Akt (Ser473) antibodies, and detected by enhanced chemiluminescence. (C) IRS-1 was immunoprecipitated from cell lysates as in panel A, but the resulting immunoprecipitates were then resolved by SDS-PAGE and immunoblotted with antiphosphotyrosine or anti–IRS-1 antibodies.