Effect of LY294002 on activation of ras after stimulation with anti-IgE Ab.

Basophils were resuspended in PAGCM (containing 1 mmol/L Ca⁺⁺) or PAG (without Ca⁺⁺) and preincubated with or without LY294002 (10 μmol/L) for 10 minutes. The cells were stimulated with or without anti-IgE (0.5 μg/mL) for 5 minutes. Reactions were stopped with the addition of ice-cold PAG, and the cells were microfuged. Clarified lysates were subjected to affinity precipitation with RBD-GST. Affinity-precipitated GTP-ras was detected by immunoblotting with anti-ras Ab. The immunoblot shown is representative of 2 separate experiments.