MyD-1 is coupled to consequent activation of PI 3-kinase, PtdCho-PLD, and sphingosine kinase. (A) U937 cells labeled with [³H]inositol were stimulated with isotype control (IgG1), LPS (100 ng/mL), anti-MyD-1 (10 μg/mL), or LPS plus anti-MyD-1 for 1 hour in the presence of 10 mM LiCl before measuring PIP₂-PLC activation by determining total [³H]inositol phosphate generation. (B) U937 cells labeled with [³H]palmitate were stimulated with isotype control (IgG1), LPS (100 ng/mL), anti-MyD-1 (10 μg/mL), or LPS plus anti-MyD-1 for 1 hour before measuring PtdCho-PLD activation by determining [3H]PtdBut generation. (C) Uninduced [³H]palmitate-labeled U937:Δp85 cells or [³H]palmitate-labeled U937:Δp85 cells induced to overexpress the dominant-negative form of p85 were stimulated with IgG1 or anti-MyD-1 (10 μg/mL) for 1 hour at 37°C. Some samples were pretreated (30 minutes) with wortmannin (5 nM or 50 nM) as indicated. (D) [³²P]-labeled U937 cells were stimulated in the presence (♦) or absence (□) of anti-MyD-1 (10 μg/mL) for the indicated times before determining levels of sphingosine-1-phosphate. (E) Uninduced [³H]serine-labeled U937:Δp85 cells or [³H]serine-labeled U937:Δp85 cells induced to overexpress the dominant-negative form of p85 were stimulated in the presence or absence of anti-MyD-1 (10 μg/mL) for 1 hour at 37°C. Some samples were pretreated (30 minutes) with wortmannin (5 nM or 50 nM) as indicated and sphingosine-1-phosphate levels determined. (F) Uninduced [³H]serine-labeled U937: Δp85 cells or [³H]serine-labeled U937:Δp85 cells induced to overexpress the dominant-negative form of p85 were stimulated in the presence or absence of anti-MyD-1 (10 μg/mL) for 1 hour at 37°C. Some samples were pretreated (30 minutes) with 0.1% butan-1-ol as indicated and sphingosine-1-phosphate levels determined. Data are expressed as means ± SD of triplicate determinations from 3 to 6 independent experiments.