Fig. 3. PKC and Src family tyrosine kinases are required for JNK activation induced by gastrin. After a 30-min preincubation with (+) or without (-) 5 µM GF109203X (GF), 1 µM Gö6983, or 10 µM PP2, CHO cells expressing the human CCKBR were stimulated for 3 min (A, C, and E) or 5 min (B and F) with 10 nM gastrin, for 20 min with 100 nM PMA (A and B), or for 30 min with 10 µg/ml anisomycin (D). A, C, D, and E, whole cell lysates were immunoblotted with an anti-phospho-Thr183 and -Tyr185 JNK antibody. B and F, after IP with an anti-JNK1 antibody, JNK activity using GST-c-Jun-(179) as an exogenous substrate was assayed as described in "Materials and Methods." Comparable amounts of proteins were detected in whole cell lysates or immunoprecipitates of all of the above-mentioned experiments (data not shown). The autoradiograms were densitometrically analyzed, and the data from at least three separate autoradiograms were plotted as fold stimulation and presented as means ± SE.