FIGURE 5.

Pyk2 phosphorylation induced by ionomycin is dependent on SFK activity. A, Pyk2 immunoprecipitates from cells stimulated with 0.5 μm ionomycin in the presence of the indicated concentration (in μm) of PP2 or the inactive analog PP3 sequentially probed with anti-phosphotyrosine then Pyk2. B, Pyk2 immunoprecipitates from AB.1 cells stimulated with the indicated concentration of ionomycin then probed with anti-phosphotyrosine, Pyk2 phosphorylation site specific antibodies then anti-Pyk2. C, AB.1 were pretreated with 10 μm PP2 or PP3 then stimulated with either 0.1 μm or 0.5 μm thapsigargin prior to lysis. Pyk2 immunoprecipitates were sequentially probed with anti-pY⁴⁰², anti-phosphotyrosine, and anti-Pyk2. D, Pyk2 immunoprecipitates from cells stimulated with cross-linked anti-CD3 antibodies (+) or cross-linking antibodies alone (−) for 10 min in the presence of the indicated concentration (in μm) of PP2 or the inactive analog PP3 sequentially probed with anti-phosphotyrosine, the indicated phosphospecific antibody followed by Pyk2. E, as in D except the cells were stimulated (+), or not (−), with plate-bound anti-CD3 for 20 min.