SYK is required for TLR4-induced cytokine secretion. (A) Levels of mRNA for IL10, IL6, and TNF in control and mutant B cells cultured for the indicated times with LPS or of cytokines secreted by B cells cultured for 16 h in the absence (−) or presence (+) of LPS. Cytokine mRNA levels were normalized to levels of Hprt mRNA. AU, arbitrary units; nd, not detectable. (B) Fraction of B cells positive for intracellular IL10 after culture for 5 h in the presence (+) or absence (−) of LPS. (C) Fraction of splenic B cells that were CD5⁺CD1d⁺. (D) Fraction of CD5⁺CD1d⁺ B cells positive for intracellular IL10 after culture for 5 h in the presence (+) or absence (−) of LPS. Graphs show mean ± SEM. Sample numbers: A, 3 (mRNA), 4 (cytokine protein, Syk^fl/+MCM), and 6 (Syk^fl/−MCM); B, 5; C, 4; and D, 5. Statistical analysis was performed using two-way ANOVA (p-values for effect of genotype; A, mRNA) and a Mann–Whitney test (A, cytokine protein; C): *, P < 0.05; **, P < 0.01.