Time-course of CD43-induced tyrosine kinase activity and presence of PYK-2 in anti-Tyr (P) immunoprecipitates. (A) NK cells were allowed to adhere for the indicated times on dishes precoated with the anti-CD43 TP1/36 MoAb. The cells were then lysed and the extracts were immunoprecipitated (IP) with the PY20 anti-Tyr(P) MoAb [IP: Tyr (P)], and kinase reactions performed as described in Materials and Methods (left panel). After the kinase reaction was performed, the major Tyr (P)-labeled bands in the cells stimulated for 60 minutes were eluted from the PY20 immunocomplex by boiling the pellet in 100 μL of a solution containing 10 mmol/L Tris, pH 7.4, and 1% SDS. Denaturated Tyr(P) proteins were then reimmunoprecipitated (r-IP) with the C-19 anti–PYK-2 antibody to confirm the presence of PYK-2 [IP: Tyr(P); r-IP: PYK-2] or with nonimmune serum control [IP: Tyr (P); r-IP: IgG; right panel]. After SDS-PAGE (7.5%) of immunoprecipitates, gels were subjected to autoradiography. The position of the major phosphorylated bands in the gel is indicated with arrowheads. PYK-2 position is indicated by an arrow. Molecular weight markers (in kilodaltons) are shown on the left side of the figure. (B) Cells were lysed and the extracts were incubated with C-19 antibody to immunoprecipitate PYK-2. Tyrosine phosphorylation levels of PYK-2 were determined by Western blot analysis with the PY20 anti-Tyr (P) MoAb.