FIG. 11.
Effect of cAMP-increasing agents on PI3-kinase activity immunoprecipitated with antibody to p85. 293 cells were preincubated for 20 min with 100 nM wortmannin prior to stimulation for 5 min with 1 μM insulin or for 30 min with 10 μM forskolin. Total cellular protein lysates were immunoprecipitated with anti-p85 antibody, and associated phospholipid kinase activities were estimated in vitro with PtdIns or PtdIns-4-P plus PtdIns-4,5-P2 as the substrate. The lower panel shows a representative thin-layer chromatogram of products of PI3-kinase in the presence or absence of wortmannin. The position of 32P-labeled PI (PIP) is indicated. The graph shown in the upper panel corresponds to the radioactivity associated with each spot as quantified by phosphorimaging. B represents the nontreated cells. IP, immunoprecipitates.