cAMP signaling inhibits PI3K activity. DHL6 parental cells were treated with 40 μM forskolin and 10 μM rolipram for 5 minutes. Cell lysates were harvested and immunoprecipitated with anti-phosphotyrosine antibody (4G10). Precipitated proteins were incubated with phosphatidylinositol (PI), phosphatidyl-4,5-biphosphate (PIP2), and [γ-32P]ATP, followed by extraction of lipids, separation by thin layer chromatography, and exposure to an x-ray film (left panel, representative assay). PI3K activity toward its major lipid substrates (PIP2) was determined by densitometrically measuring the amounts of the phosphatidylinositol 3,4,5-trisphosphate (PIP3) produced in 3 independent reactions (right panel). Fold of inhibition (right panel) was determined by comparing the PIP3 intensity in forskolin/rolipram-treated and untreated cells and reported as the mean ± SD.