Stimulation of human U937 monocytes with anti-MyD-1 stimulates PI 3-kinase. (A) Uninduced [32P]-labeled U937:Δp85 cells were stimulated in the presence (▪) and absence (□) of anti-MyD-1 (10 μg/mL) for the indicated times at 37°C. Pretreatment (30 minutes) with wortmannin (1 nM) had little effect on basal PIP₃ levels (▵) but suppressed anti-MyD-1-stimulated PIP₃ production (▴). Similar anti-MyD-1 stimulations were carried out on cells induced to overexpress the dominant-negative form of p85. IPTG-induced U937:Δp85 cells were stimulated in the presence (•) and absence (○) of anti-MyD-1 (10 μg/mL) for the indicated times at 37°C. Lipids were extracted and [32P]PIP₃ production was identified by TLC and quantified by liquid scintillation counting. Data are presented as means ± SD, n = 3, and are representative of 5 independent experiments demonstrating coupling of MyD-1 to PI 3-kinase. (B) Anti-SHP-2 immune complexes from U937 cells were assessed for p85 expression, tyrosine phosphorylation, and SHP-2 expression.