FIGURE 4.

Calcium-induced Pyk2 phosphorylation does not require calmodulin. A, AB.1 were stimulated with 1 μm thapsigargin for 10 min then lysed. Immunoprecipitates were prepared with antibodies specific for either the N terminus (NT) or C terminus (CT) of Pyk2 or with antibodies specific for calmodulin. A single membrane was first probed with anti-phosphotyrosine then stripped and simultaneously probed for Pyk2 and calmodulin. B, AB.1 cells were pretreated with the indicated concentration of the calmodulin inhibitor W7 or its inactive analog W5 for 20 min prior to stimulation of the cells for 10 min with 0.5 μm thapsigargin. Pyk2 immunoprecipitates were prepared then sequentially probed with anti-phosphotyrosine then anti-Pyk2. C, Pyk2 immunoprecipitates from cells stimulated with ionomycin in the presence of increasing concentrations of KN-62 probed with anti-phosphotyrosine then anti-Pyk2.