PMN interaction with activated platelets stimulates αMβ2-dependent Pyk2 phosphorylation. (A) Human PMNs were preincubated with the function-blocking anti–β2-integrin mAb IB4 or with an irrelevant mouse monoclonal antibody for 15 minutes in ice. Thrombin-activated fixed platelets were preincubated with control or the anti–P-selectin mAb WAPS for 15 minutes at room temperature. PMNs and platelets were stirred at 1000 rpm at 37°C for 3 minutes. Pyk2 was immunoprecipitated from total cell lysates, and immune complexes were analyzed for phospho-Pyk2 by immunoblotting with PY99, and then the blots were reprobed with anti-Pyk2 antibody to detect total Pyk2. The bars report the ratio between optical density of PY99 andPYK2 (ie, phospho-Pyk2/total Pyk2). The figure is representative of results obtained in 3 different experiments. (B) PMNs from wild-type or αM^−/− mice were stirred in the presence of fixed-activated wild-type or P-selectin^−/− platelets at 37°C for 3 minutes. The interactions were stopped by the addition of an equal volume of reduced sample buffer, and samples were processed for immunoblot analysis as described for panel A. The bars indicate the ratio between optical density of PY99 and PYK2 (phospho-Pyk2/total Pyk2). The figure is representative of results obtained in 2 different experiments performed with PMNs isolated from pooled bone marrow obtained from 5 wild-type or 5 knock-out mice.