Measurement of total and phosphotyrosine-associated PI3K activity. Lysates from 1 × 106 cells from WT and KO cells were immunoprecipitated with pan85 antibody (A) or lysates from 5 × 106 cells with pTyr antibody (B). Imatinib was included for 15 minutes prior to cell lysis in the indicated sample (v-ABL WT). Immune complexes were subjected to an in vitro PI3K assay using phosphatidylinositol as substrate and the products resolved by thin-layer chromatography (Ori indicates origin). Radioactivity in the phosphatidylinositol-3-phosphate product (PIP, indicated by arrow) was quantitated by phosphoimager and graphed below. Similar results were obtained in 3 independent experiments of 2 different cell lines per genotype. The specificity of the immune complex kinase assays was verified in control experiments showing that kinase activity was completely blocked by in vitro treatment with wortmannin (50 nM), a selective PI3K inhibitor (not shown).
