Figure 5
BTK exerts an activating and PI3K a permissive role on ERK1/2 activation in MCs stimulated by suboptimal FcεRI crosslinking. (a,b) WT (+) and Btk−/− (−) PMCs were left untreated (con) or stimulated for 1 and 5 min or (b) for 5 min with the indicated amounts of Ag (DNP-HSA [DNP]). (c) WT BMMCs were pre-treated for 30 min with 1 µM of the PLC-inhibitor U73122, its inactive compound U73343, or their vehicle (DMSO) and subsequently stimulated for 1 min with suboptimal (0.5 ng/ml) and optimal (20 ng/ml) concentrations of Ag. (d) WT and Btk−/− BMMCs were left untreated (con) or either stimulated for 1 min in parallel with 2 and 20 ng/ml Ag and increasing amounts of PMA (ng/ml) or vehicle (DMSO). Between lanes 12 and 13, no lysate was applied. (e) WT and Btk−/− BMMCs were pre-treated for 20 min with 100 nM Wortmannin (WM) before stimulation with 1 and 20 ng/ml Ag for 1 and 5 min. (f,g) WT BMMCs were pre-treated for 30 min with 0.3 µM Ibrutinib (f) or 20 min with 100 nM Wortmannin (g) before stimulation for 1 and 5 min with 2 µg/ml IgE (SPE-7). (a–g) Whole cell-lysates were subjected to immunoblot analysis with the respective antibodies against P-PKB (Ser473), P-ERK1/2 (Thr202/Tyr204), and p85 or GAPDH as loading controls. Comparable results were obtained in at least three experiments with different PMC or BMMC cultures. Fine vertical lines were inserted to allow for better discrimination of cells/conditions. (h) Model integrating the positive and negative effects of BTK and PI3K, respectively, on activation of ERK1/2. KO phenotypes and pharmacological activators/inhibitors are highlighted in fawn.
