Syk expression and activation in wild-type fetal liver–derived eosinophils.

(A) Expression of Syk in lysates of wild-type and Syk^−/−fetal liver–derived eosinophils. Whole-cell lysates were subjected to SDS-PAGE and Western blotted with polyclonal rabbit anti-Syk antibody (Santa Cruz). (B) Starved mature eosinophils from 8-day-old cultures were stimulated with either 50-ng/mL IL-5 for 15 minutes (IL-5) or with anti-CD16/CD32 2.4G2 monoclonal antibody (2 μg/mL) for 30 minutes on ice and further cross-linking with goat antirat polyclonal antibody for 15 minutes at 37°C (Fc). The cell lysates were immunoprecipitated with a polyclonal anti-Syk antibody, and the immunoprecipitates were subjected to SDS-PAGE and Western blotted with polyclonal rabbit anti-Syk antibody for determination of Syk enzyme levels. (C) Lysates from stimulated cells were immunoprecipitated with a polyclonal anti-Syk antibody or with rabbit IgG (nonspecific, NS) and subjected to an in vitro kinase assay. Data are expressed as mean ± SEM of 4 independent experiments. Significance of differences of Syk activity was determined by the Student t test, andP < .05 (*) or P < .005 (**) was considered significant.