Activation of protein kinases by TLR ligands and antigen and effects of MAP kinase inhibitors on TNFα production in MC/9 cells. IgE-primed MC/9 cells were stimulated or not (NS) for the periods indicated with 100 ng/mL LPS, 1 μg/mL P3C, and 20 ng/mL antigen (Ag), individually or in combination. (A) IRAK1 was immunoprecipitated and assayed for kinase activity by incubation with [γ-³²/P]ATP and myelin basic protein (MBP). The product, ³²P-labeled MBP, was separated and detected by electrophoresis and autoradiography. (B) Immunoblots were prepared also from cell extracts and then probed with antibodies that detected either the indicated activated phosphorylated protein kinase or the protein itself. The blots are representative of blots from at least 3 separate experiments. (C) The relative densities of the doubly phosphorylated (Thr183/Tyr185)–JNK band were determined by densitometry. The values are the mean ± SEM of 4 experiments that were terminated 15, 30, and 60 minutes after addition of ligand(s). (D) IgE-primed MC/9 cells were incubated with vehicle, 25 μM SP 600125, 10 μM SB 203580, or 30 μM PD 98059 for 1 hour before addition of 100 ng/mL LPS, 1 μg/mL P3C, and 20 ng/mL antigen, individually or in combination. TNFα was assayed by ELISA 3 hours thereafter. ND indicates not detectable. Values are mean ± SEM of 4 cultures and are from 1 of 2 identical experiments.