Effect of Erb-A on PI3K lipid kinase activity. (A) Triton-soluble fractions of resting platelet lysate were immunoprecipitated with equal amounts of either PI3K antibody (lanes 5 through 10) or an isotype-matched antibody (lane 4). The washed immune complexes were subjected to a “cold” in vitro kinase assay in the presence of ATP/Mn²⁺, and in the absence or presence of the indicated concentrations of Erb-A. Control reactions containing no Erb-A were incubated with either buffer alone (lanes 4 and 5) or ethanol/buffer vehicle (lane 6). The phosphorylated immune complexes were washed and incubated for 10 minutes in the presence of γ-³²P-ATP, Mg²⁺, and PI(4,5)P₂substrate. Radiolabeled phosphoinositides were extracted and separated by TLC as described in Materials and Methods. Migrations of authentic PI(4,5)P₂, PI(4)P, or PI standards are indicated by outlined circles. PI; phosphatidylinositol. (B) Direct quantitation of radiolabeled spots from lanes 6 through 10 in (A). Each bar represents the fold-increase of PI(3,4,5)P₃ formed (net counts per minute) relative to the buffer-only control (A, lane 5). Results are presented as the mean of three experiments ± standard error.