Effect of LY294002 on phosphorylation of syk and shc and association of Grb2 and shc after stimulation with anti-IgE Ab.

Basophils were preincubated with DMSO (1:10 000 dilution) or LY294002 (10 μmol/L) for 10 minutes and were stimulated with 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 immunoprecipitated with anti-syk (A), anti-shc (B), or anti-Grb2 (C). The immunoprecipitated proteins were subjected to Western blot analysis with the indicated Abs, as described in “Materials and methods.” The same membranes were stripped and reblotted with the indicated Ab. The anti-syk (A), anti-shc (B), and anti-Grb2 (C) blots indicate essentially equal protein loading. Each Western blot shown is representative of 2 separate experiments.