AKO mast cells were refractory to IgE/antigen stimulation. (A) Ca²⁺-mobilization and influx. BMMCs of WT and AKO were sensitized with IgE (E-C1) for 16 hours and then loaded with the Ca²⁺ indicator Fluo-3-AM and Fura red-AM. Ca²⁺ release was elicited by stimulation with 10 μg/mL OVA (Ag) first in Ca²⁺-free conditions, followed by shifting to Ca²⁺ (2 mM)-containing buffer 4 minutes later. The resulting changes in intracellular calcium were monitored over time. Data are representative of 3 independent experiments. (B) BMMCs were sensitized with E-C1 and then stimulated with 10 μg/mL OVA for the indicated time. Whole-cell lysates were analyzed by western blot. Data are representative of 3 independent experiments. (C) The levels of degranulation, IL-13 (D), and LTC₄ (E) in WT or AKO mast cells sensitized with IgE (E-C1) for 16 hours and stimulated with OVA for 30 minutes for degranulation and LTC₄ or 6 hours for IL-13 measurement. *P < .05. Data are representative of 5 independent experiments.