Time course of changes in the levels of PtdIns(3,4,5)P₃ in fMLP-stimulated, TNF-α-primed mouse neutrophils. Neutrophils derived from the bone marrows of the indicated strains of mice were labeled with [³²P]Pi in the presence of 500 U/mL TNF-α, as described in “Materials and methods.” Neutrophils were washed, warmed to 37°C over 5 minutes, and challenged with 10 μM fMLP (▴) or vehicle (▵) for the indicated times. Dotted lines without symbols indicate values for fMLP-stimulated wild-type (WT) cells. Incubations were terminated, and [³²P] phosphoinositides were quantified as described in “Materials and methods.” Data shown are for the levels of [³²P] PtdIns(3,4,5)P₃ (mean ± SD; n = 3 collected from separate preparations of neutrophils; where not shown, errors lie within the symbols). Analogous data showing the levels of the other [³²P] phosphoinositides measured are described in Figure S3.