Regulation of LMW-PTP in FcγRIIA-stimulated platelets. (Ai). Platelets were stimulated in a lumiaggregometer by clustering of FcγRIIA (2 μg/mL IV.3 monoclonal antibody and 30 μg/mL antimouse F(ab′)₂ fragments) for increasing times. Upon immunoprecipitation with the specific antibody against LMW-PTP, or with unrelated IgG used as negative control, the level of tyrosine phosphorylation was investigated by immunoblotting, as indicated on the right. The percentage of platelet aggregation measured in each stimulated sample is reported on the bottom. (ii) Lysates from v-src–transformed NIH3T3 cells were immunoprecipitated with anti–LMW-PTP antibody (lane 1) or with control, unrelated IgG (lane 2). Immunoprecipitated proteins were analyzed by immunoblotting with antiphosphotyrosine antibody (P-Tyr, top panel) and with anti–LMW-PTP antibody (bottom panel). (B) Platelet stimulation by clustering of FcγRIIA was performed in a lumiaggregometer, and was stopped at the indicated times. Upon cell lysis, the intracellular cytoskeleton was isolated as Triton-X-100–insoluble material. The association of LMW-PTP with the cytoskeleton was analyzed by immunoblotting with anti–LMW-PTP polyclonal antibodies. The figure also shows the percentage of platelet aggregation measured in each sample. (C) Cytosol (cyt) and membrane-rich fractions (memb) were separated from platelets stimulated by clustering of FcγRIIA for the indicated times. The subcellular redistribution of LMW-PTP was investigated by immunoblotting with the specific polyclonal antibodies. All these experiments have been repeated at least 3 times.