Activation of the Ins-R/Cbl/C3G pathway occurs 5 min after insulin stimulation. Skeletal muscle cells were differentiated for 5 days and treated with or without insulin (160 nM) for 2, 5, and 20 min. Cell lysates were immunoprecipitated with anti-Ins-Rβ (A), anti-Cbl IgGs (B), anti-CrkII IgGs (B), and anti-C3G IgGs (B, C), and immunoprecipitates subjected to immunoblot analysis using anti-caveolin-3 IgGs (A), anti-flotillin-1 IgGs (A, B), and anti-phosphotyrosine IgGs (C). Total caveolin-3, flotillin-1, Cbl, CrkII, and C3G protein expression was not affected by insulin (data not shown).