Both KL and antigen stimulate PLCγ₁ phosphorylation; production of inositol 1,4,5-trisphosphate; and calcium mobilization. Human mast cells were incubated overnight in the presence of anti–4-hydroxy-3-nitrophenylacetyl–IgE in the absence of KL and IL-6. Cells were not stimulated or were stimulated with 100 ng/mL KL, antigen (Ag), or both in combination for the periods indicated to assess the extent of tyrosine phosphorylation of PLCγ₁ (p-PLCγ₁) by immunoblotting (A); intracellular concentrations of inositol 1,4,5-trisphosphate (IP₃; B); and concentration of intracellular-free Ca²⁺ (C) as described in “Materials and methods.” Data are from 1 of 3 representative experiments (A,C) or are the mean ± SEM of values from 3 separate experiments (B). The data in panel C indicate the ratio of fluorescence at 510 nm when Fura-2–loaded cultures in multi-well plates were excited at 340 nm and 380 nm and are the average value from 3 cultures.