BCR is required for TLR4-induced activation of SYK. (A) Immunoblot of lysates from wild-type B cells stimulated for various times with LPS (left) or LAS (right). Blots were probed with antibodies to pSYK and SYK. Graphs show mean ± SEM levels of pSYK normalized to SYK or tubulin and then to unstimulated cells (time 0), quantitated from multiple immunoblots. n = 4 (LPS) and 3 (LAS). (B–E) Immunoblots of lysates from IgM⁺ or IgM⁻ B cells (B–D) or Myd88^+/+ or Myd88^−/− B cells (E) stimulated with LPS (B and E), CD40L (C), or LAS (D) for the indicated times. Blots were probed with antibodies to pSYK, pAKT, pERK, SYK, ERK2, and IκBα. Graphs show mean ± SEM levels of pSYK, pAKT, pERK, and IκBα normalized to SYK or ERK2, and then to the levels in unstimulated control B cells (time 0), quantitated from multiple immunoblots. Minimum number of repeats per time point: B, 9 (pSYK except 3 for 15 min), 8 (pAKT), 7 (pERK), and 5 (IκBα); C, 3 (pERK), 3 (IκBα), and 2 (pAKT); D, 3 (pSYK), 4 (pERK), and 4 (IκBα); E, 9 (pSYK) and 7 (pAKT); 2 (pERK and IκBα in WT), 4 (pERK and IκBα in Myd88^−/−). Numbers on righthand edge of all blots indicate molecular mass makers (kilodaltons). Statistical analysis was performed using a Mann–Whitney test (A and B, pAKT) or two-way ANOVA (all other graphs; p-values for effect of genotype): *, P < 0.05; ***, P < 0.001; ****, P < 0.0001.