FIGURE 3.

Regulation of insulin-induced PI3K activity and association with IRS1 by PKC activation. A, effect of constitutively active p110/PI3K (myr-P110A) on p-Akt. BAEC, transfected with GFP or constitutively active p110/PI3K plasmid, were treated with insulin or PMA. p110α, p-Akt, and t-Akt were assessed by immunoblot analysis in three separate experiments (n = 3). B, effect of insulin and PMA on PI3K activity associated with IRS1. Left, representative thin layer chromatography with arrows point to ³²P incorporation into phosphatidylinositol (PIP). Right, bar graph shows densitometrical quantitation of ³²P-phosphorylated phosphatidylinositol (n = 3; *, p < 0.001). C, effect of PKC activation on IRS1-associated p85α/β in IRS1-overexpressed cells. Top, immunoprecipitation (IP) with anti-IRS1 antibodies followed by immunoblotting (IB) with indicated antibodies. Bottom, densitometry quantitation of p85α/β to IRS1 levels (n = 3; *, p < 0.001; #, p < 0.005). D, effect of PKC activation on the amount of p85-associated IRS1 in IRS1-overexpressed cells. Top, immunoprecipitation with anti-p85α/β antibodies followed by immunoblot with indicated antibodies are shown. Bottom, densitometry quantitation of IRS1 to p85α/β signal ratio are shown (n = 3; *, p = 0.001; #, p < 0.001).