Identification of platelet substrates for LMW-PTP. FcγRIIA, Syk, LAT, and PLCγ2 were immunoprecipitated from platelets stimulated by clustering of FcγRIIA (2 μg/mL IV.3 monoclonal antibody and 30 μg/mL antimouse F(ab′)2 fragments), while FcR γ-chain was immunoprecipitated upon platelet stimulation with 100 ng/mL convulxin. Immunoprecipitated proteins were separated by SDS-PAGE and transferred to nitrocellulose. Membranes were then incubated in the absence or presence of purified LMW-PTP for 30 minutes (A). Alternatively, the immunoprecipitated proteins immobilized on protein A–Sepharose were incubated in the absence or presence of purified LMW-PTP for 30 minutes and subsequently separated by SDS-PAGE (B). The level of tyrosine phosphorylation of the immunoprecipitated proteins was then evaluated by immunoblotting with antiphosphotyrosine antibody, and, upon stripping, each membrane was reprobed with the same antibody used for the immunoprecipitation, as reported on the right of each panel. In panel C, a quantification of the residual phosphorylation of the analyzed substrates after incubation with LMW-PTP on nitrocellulose (□) or in solution (■) is reported. The level of phosphorylation of each single protein in the absence of LMW-PTP is taken as 100%. The data are reported as mean plus or minus SD of 3 to 5 different experiments for each protein.
