Fig. 2.
Jun kinase activity increases during PC12 differentiation and when differentiated PC12 cells are deprived of NGF.A, Control immune complex kinase assays were performed with extracts from quiescent (−UV) and UV-treated (+UV) HeLa cells as described in Materials and Methods. One hundred micrograms of extract were used for each immunoprecipitation. Competition with the c-Jun δ and Bax peptides was performed as described in Materials and Methods. The products of the Jun kinase assay were separated on a 10% SDS-polyacrylamide gel, which was fixed and dried, and autoradiography was performed. The position of phosphorylated GSTc-Jun[1–169] is shown. B, Jun kinase activity increases during PC12 differentiation. PC12 cells were treated with NGF, and extracts were prepared immediately after NGF addition (time 0) or 7 d later (7d). Jun kinase assays were performed using 100 μg of extract, as described in Materials and Methods. A representative result is shown. On average (7 experiments), Jun kinase activity increased fourfold during PC12 differentiation.C, Jun kinase is activated when differentiated PC12 cells are deprived of NGF. Differentiated PC12 cells were refed with defined medium lacking NGF, which had been supplemented with anti-NGF antibody. Extracts were made at the times indicated, and immune complex kinase assays were performed, using 100 μg of extract/immunoprecipitation. The resulting autoradiograph was scanned on a densitometer to determine the relative levels of Jun kinase activity at different times. The level of Jun kinase activity at time0 was set as 100%. The results shown are the average values for three independent experiments. Error bars indicate SE.