Effects of PI3K inhibitors on PtdIns(3,4,5)P₃ accumulation in fMLP-stimulated, TNF-α-primed human neutrophils. Human neutrophils were labeled with [³²P]Pi in the presence of 200 U/mL TNF-α, as described in “Materials and methods.” Neutrophils were washed and warmed for 5 minutes at 37°C with the indicated concentration of inhibitor and then challenged with 100 nM fMLP for either 6 seconds or 60 seconds. Incubations were terminated, and [³²P]phosphoinositides were quantified as described in “Materials and methods.” Data shown are for the accumulation of PtdIns(3,4,5)P₃ expressed as a percentage of the value with no inhibitor; data are mean ± SD or range (n = 2-5) collected from separate preparations of neutrophils (where not shown, errors lie within the symbols). Analogous dose curves describing the effects of the inhibitors on the other [³²P] phosphoinositides are shown in Figure S2.