FIG. 5.

FcɛRI-induced tyrosine phosphorylation and enzymatic activity of Y342F mutant Syk. (A) FcɛRI-induced tyrosine phosphorylation of Syk. Cells were cultured overnight with antigen specific IgE and either nonstimulated or stimulated with antigen (0.1 μg/ml) (− or + Ag) for 20 min. Lysates were immunoprecipitated with rabbit anti-Syk antibody. The immunoprecipitates were analyzed by immunoblotting with antiphosphotyrosine (4G10) and mouse anti-Syk antibody. (B) In vitro kinase assay of Syk. Syk was immunoprecipitated from lysates of either nonstimulated or stimulated cells as in panel A and then incubated with [γ-³²P]ATP in a protein kinase assay. The proteins were separated by SDS-PAGE, electrotransferred, and visualized by autoradiography. The membranes were then blotted with anti-Syk antibody. The results shown are representative of at least three independent experiments.