Fig. 6.

Rapid and specific activation of SAPK/JNK by glutamate. A, Immune-complex kinase assay for SAPK/JNK activity at different times after treatment of primary striatal cultures with glutamate (100 μm). The bands indicate c-Jun(1–135)-GST substrate (∼40 kDa) that was phosphorylated by anti-SAPK/JNK immunoprecipitates before separation in a polyacrylamide gel. The last lane shows the reaction product using cells that were treated with an additional 100 μmglutamate for the last 45 min of a 6 hr treatment. Fold increases are shown above each lane. B, On theleft, a Western blot for SAPK/JNK performed on lysates of untreated striatal cultures and those treated with glutamate (100 μm) for 1 or 2 hr. On the right, an in-gel kinase assay on anti-SAPK/JNK immunoprecipitates from striatal cultures. The phosphorylation of c-Jun(1–135)-GST substrate, which had been incorporated into the polyacrylamide gel, was performed in the gel after separation of immunoprecipitate components. When parallel in-gel kinase assays were performed without c-Jun substrate polymerized into the gel, kinase activity was reduced >100-fold (data not shown). The positions of flanking molecular weight markers are indicated forA and B. C, Comparison of immune-complex kinase assays for SAPK/JNK activity (top graph; using c-Jun(1–135)-GST as substrate) and p38 activity (bottom graph; using myelin basic protein as substrate) in immunoprecipitates of striatal cultures. In B(right) and C, treatments lasted 45 min with glutamate (Glu), dopamine (DA), and carbachol (Carb) at 100 μm; forskolin (Fors) at 10 μm; and mannitol (Mann) at 300 mm. The data inA and B were typical of at least three experiments. The data in C were pooled from two experiments, with n = 6 (except for the forskolin group, in which n = 3); * greater than control, with p < 0.05; ** less than control (by 10%), with p < 0.05.