Activation of PI3K by tyrosine kinases in human neutrophils. (A-C) Human neutrophils (1 × 10⁷) were primed with 200 U/mL TNF-α for 30 minutes (unless otherwise indicated), then challenged with 1 μM fMLP or vehicle for 30 seconds. Detergent-soluble lysates were immunoprecipitated with antiphosphotyrosine antibody, and immunoprecipitates were assayed for PI3K activity, as described in “Materials and methods.” Data shown are expressed as a percentage of basal activity and are mean ± SEM of at least 3 experiments performed in at least duplicate. ^**P < .001 compared with no TNF/fMLP; Student t test. Where appropriate, PI3K inhibitors (15 nM TGX-221, 1 μM IC87114, 80 nM AS-252424) or DMSO controls were preincubated with immunoprecipitates for 5 minutes before measurement of PI3K activity. (D) Human neutrophils were labeled with [³²P]Pi in the presence of 200 U/mL TNF-α, as described in “Materials and methods.” Washed cells were warmed for 5 minutes at 37°C with either 25 μM LFM-A13, 20 μM PP1, or their vehicle (0.01% DMSO) and then challenged with 100 nM fMLP for either 6 or 60 seconds. Incubations were terminated, and [³²P] phosphoinositides were measured as described. Data shown are for the levels of [³²P]PtdIns(3,4,5)P₃ (mean ± SD; n = 3) from a single preparation of neutrophils.