Fig. 2. Glutamate induces activation and tyrosine phosphorylation of PKC in HT22 cells. (A) HT22 cells were incubated with 5 mM glutamate (Glu) for the indicated times. Cell extracts were subjected to immunoprecipitation with anti-PKC. An in vitro immune complex kinase assay were performed using histone H1 as a substrate. The immunoprecipitates were also analyzed by immunoblotting with anti-PKC. (B) HT22 cells were incubated with 5 mM glutamate for the indicated times. Cells were then harvested, and immunoprecipitation of PKC was performed with an anti-PKC antibody as described in the Materials and Methods. Membranes were immunoblotted with anti-phosphotyrosine antibody (pTyr) and/or anti-PKC antibodies. The relative kinase activities and levels of tyrosine phosphorylation of PKC were quantified by densitometric analyses, and normalized to the level of total PKC (lower panel in A and B). Data represent an average fold increase as compared with controls (mean ± s.d.). ^*P<0.05 and ^**P<0.01, values compared with zero time. (C) HT22 cells were incubated with 5 mM glutamate for the indicated times, and the cleavage of PKC was determined by western blotting. The 40 kDa cleaved form is marked by an arrow. Results were reproducible, and blots shown are representative of three separate experiments. CL, cleaved form; FL, full-length form; HC, heavy chain.