Protein tyrosine phosphorylation in HuMCs and degranulation following FcϵRI aggregation and SCF challenge. Total cellular protein phosphorylation was assessed in panels A-B by sensitizing HuMCs overnight with 1000 ng/mL NP-IgE, and then triggering for 5 minutes with (A) the indicated concentrations of NP-BSA or (B) 100 ng/mL NP-BSA for the indicated periods of time. Membranes were probed with an antiphosphotyrosine antibody. (C-D) For degranulation experiments, HuMCs were sensitized overnight with either 1000 ng/mL (•) or 100 ng/mL (○) NP-IgE and then triggered for 30 minutes (C) with the indicated concentrations of NP-BSA or (D) with NP-BSA (100 ng/mL) for the indicated periods of time. ▾ indicates no sensitization. (E) HuMCs were cultured overnight in the absence of SCF but in the presence of NP-IgE (100 ng/mL). The cells were then challenged with SCF either in the absence (○) or presence of NP-BSA: (▴), 0.01 ng/mL; (▾), 0.1 ng/mL; (♦), 1 ng/mL; (▪), 10 ng/mL; and (•), 100 ng/mL. (F) HuMCs were challenged for the indicated times with SCF (100 ng/mL) in the absence of NP-BSA prior to probing for phosphotyrosine as described in “Materials and methods.” The degranulation experiments are means ± SEM of n = 2 to 5 experiments conducted in duplicate, and the blots are representative of at least n = 3.