Analysis of Syk tyrosine phosphorylation in DAMI cells. (A) The 3 different clones of DAMI megakaryocytic cells (wild type, S2C, and PTP13) were stimulated by clustering of FcγRIIA (2 μg/mL IV.3 monoclonal antibody and 30 μg/mL antimouse F(ab′)2 fragments) for the indicated times. The tyrosine kinase Syk was then immunoprecipitated and subjected to immunoblotting analysis with antiphosphotyrosine antibody and, upon stripping of the nitrocellulose, with anti-Syk antibody, as indicated on the right. (B) Comparison of the kinetics of Syk phosphorylation in wild-type (■), S2C (•), and PTP12 (▴) DAMI cells stimulated by clustering of FcγRIIA, as evaluated by densitometric analysis of the results of 3 different experiments. Results are reported as means plus or minus SD and have been normalized based on an experiment in which samples at a single time point for all the 3 clones have been analyzed on the same immunoblot.
