Effect of wortmannin or LY294002 on protein kinase activity of PI3K. (A) PI3K was immunoprecipitated from, post-130,000g, cytosolic fractions of platelet lysates (lanes 2 through 8). Lane 1 consisted of Protein G beads alone incubated with lysate. The washed immune complexes were incubated for 20 min with either kinase buffer alone (lanes 1 to 2), buffer/dimethylsulfoxide vehicle (lane 3), or the indicated wortmannin (Wort) concentrations (lanes 4 through 8). (B) Same as in (A) except washed immune complexes were incubated with either kinase buffer alone (lanes 1 and 2), buffer/ethanol vehicle (lane 3), or the indicated LY294002 (LY) concentrations (lanes 4 through 8), and lane 1 represents an IgG₁ control antibody immunoprecipitate. Incubations were then subjected to an in vitro kinase assay as described in Fig 1.³²P-labeled proteins were eluted, separated by SDS-PAGE and detected by autoradiography. Molecular mass markers in kilodaltons are indicated at the left.