Figure 7. Pyk2 is a component of P110 and undergoes tyrosine phosphorylation in an Src-kinase- and integrin-dependent manner.

(A) Lysates from unstimulated or E-selectin-activated PMN were analysed by Western blot (w.b.) with anti-phosphotyrosine antibody PY99. After stripping, the same samples were re-probed with anti-Pyk2 antibody. (B) Lysates from unstimulated or E-selectin-activated PMN were subjected to two runs of immunoprecipitation with non-immune goat antiserum (control) or with anti-Pyk2 antibody (Pyk2-depleted) (w.b., Western blot). Tyrosine-phosphorylated proteins in the remaining supernatants were then immunoprecipitated with PY99 anti-phosphotyrosine antibody (Pyk2-precipitated). After stripping, the same samples were re-probed with anti-Pyk2 antibody. In control samples, Pyk2 can be detected in PY99 immunoprecipitates and corresponds to a clear-cut tyrosine-phosphorylated protein in the upper region of P110. This protein is not detectable in Pyk2-depleted PY99 immunoprecipitates. (C) PMN untreated or pretreated with DMSO, 10 μM PP2 or 20 μg/ml IB4 were incubated under stirred conditions for 3 min in the absence or in the presence of soluble E-selectin (E-sel) or CHO-E (5:1 PMN/CHO-E). Pyk2 was immunoprecipitated from cell lysates, and immune complexes were first analysed by Western blot (WB) with PY99 and then re-probed with anti-Pyk2 antibody. Bars indicate absorbances of PY99 Western blots. The Figure shows the results from one representative of three different experiments. (D) PMN from C57BL6 (wild-type), or from double hck^−/−fgr^−/− or triple hck^−/−fgr^−/−lyn^−/− mice, were incubated in the absence or in the presence of CHO-E for 3 min at 37 °C with stirring at 1000 rev./min. Pyk2 was immunoprecipitated from cell lysates, and immune complexes were first analysed by Western blot (WB) with PY99 and then re-probed with anti-Pyk2 antibody. Bars indicate the ratios between the absorbances of PY99 and Pyk2 Western blots. Values are from one representative of two different experiments.