Tyrosine phosphorylation of p85 after stimulation with anti-IgE Ab and FMLP, and the effect of LY294002 or PP1.

Basophils were stimulated with or without anti-IgE (0.5 μg/mL) (A) or FMLP (1 μmol/L) (B) for the times indicated. (C) Basophils were pretreated with or without DMSO, LY294002 (10 μmol/L), or PP1 (10 μmol/L) for 10 minutes and stimulated by anti-IgE Ab for 5 minutes. Reactions were stopped with the addition of ice-cold PAG, and the cells were microfuged. Clarified lysates were immunoprecipitated with anti-p85 Ab. The immunoprecipitated proteins were subjected to Western blot analysis with antiphosphotyrosine Ab as described in “Materials and methods.” The same membranes were stripped and reblotted with anti-p85 Ab. The anti-p85 blot indicates essentially equal protein loading. Each Western blot shown is representative of 2 separate experiments.