FIGURE 3.

The calcium requirement for TCR-stimulated Pyk2 phosphorylation depends on the method of stimulation. AB.1 were left unstimulated (−) or stimulated (+) with cross-linked anti-CD3 (A) or plate-bound immobilized anti-CD3 (B) for the indicated time, in the presence or absence of 4 mm EGTA. The unstimulated control (−) for the cross-linked anti-CD3 contains secondary cross-linking antibody alone and for the plate-bound stimulation the cells were plated on BSA-blocked plastic. Cells were lysed and Pyk2 immunoprecipitates were probed with anti-phosphotyrosine followed by anti-Pyk2. AB.1 cells were pretreated with either 30 μm BAPTA or vehicle control (DMSO) for 15 min on ice then stimulated with cross-linked anti-CD3 (C) or immobilized anti-CD3 (D) for the indicated time. Pyk2 immunoprecipitations were performed and probed for phosphotyrosine then Pyk2.