Regulation of FcγRIIA tyrosine phosphorylation by LMW-PTP in DAMI megakaryocytic cells. (A) The expression of LMW-PTP and FcγRIIA in wild-type DAMI cells, as well as in the clones S2C and PTP13, was analyzed on 20 μg of whole cell lysates by immunoblotting with specific antibodies as indicated on the right. (B) Wild-type, S2C, and PTP13 DAMI cells were stimulated by clustering of FcγRIIA (2 μg/mL IV.3 monoclonal antibody and 30 μg/mL antimouse F(ab′)2 fragments) for increasing times. Upon cell lysis, FcγRIIA was immunoprecipitated and the level of tyrosine phosphorylation of the receptor was analyzed by immunoblotting with antiphosphotyrosine antibody, as indicated on the right. Upon stripping, the efficiency of the immunoprecipitation was verified by reprobing the membranes with anti- FcγRIIA antibody. (C) Comparison of the kinetics of FcγRIIA tyrosine phosphorylation in wild-type (■), S2C (•), and PTP12 (▴) DAMI cells stimulated by clustering of FcγRIIA. Data are the means plus or minus SD obtained from the densitometric analysis of 3 to 5 different immunoblots similar to those reported in panel B, 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. The asterisk indicates that the differences between the S2C or the PTP13 clones and wild-type cells are statistically significant (P < .05).
