SYK transduces TLR4 signals leading to activation of ERK and AKT but not NF-κB. (A–C) Immunoblots of lysates from control and mutant B cells stimulated with LPS (A), CD40L (B), or LAS (C) for the indicated times. Blots were probed with antibodies to phospho-Ser473 AKT (pAKT), phospho-Thr202/Tyr204 ERK1/ERK2 (pERK), SYK, IκBα, and ERK2. Graphs show mean ± SEM levels of pAKT, pERK, and IκBα normalized to ERK2 and then to the levels in unstimulated control B cells (time 0), quantitated from multiple immunoblots. Minimum number of repeats per time point: A, 3 (pAKT), 9 (pERK), and 3 (IκBα); B, 3 (pAKT), 7 (pERK), and 6 (IκBα); C, 4 (pERK) and 4 (IκBα). Numbers on righthand edge of all blots indicate molecular mass makers (kilodaltons). Statistical analysis was performed using two-way ANOVA (p-values for effect of genotype): *, P < 0.05; ****, P < 0.0001.