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Fig. 3. Inhibition of PKC activity suppresses glutamate-induced ERK1/2 activation. (A) HT22 cells were incubated with 5 mM glutamate (Glu) for 7 hours in the presence or absence of 5 µM rottlerin (Rott). (i) Phosphorylation of ERK1/2 in whole cell extracts was examined by western blot analysis using a phospho-ERK1/2 (pERK1/2)-specific antibody, and (ii) PKC activity was determined by immune complex kinase assay as described in the Materials and Methods. Results were reproducible, and blots shown are representative of three separate experiments. (B) HT22 cells were transfected with either a mock vector or a dominant-negative mutant (DN) of PKC (PKC DN) vector. After 12 hours, the cells were incubated with or without 5 mM glutamate for 7 hours. (i) Phosphorylation of ERK1/2 was examined by western blot analysis using a phospho-ERK1/2-specific antibody, and (ii) PKC activity was determined by immune complex kinase assay. Relative phospho-ERK1/2 levels shown in the columns below were determined from densitometric scanning of enhanced chemiluminescence-exposed film. Each bar is a mean ± s.d. value of three independent experiments normalized by arbitrarily setting the mock-transfected cell densitometrics to 1. (C) (i) Representative photomicrographs of apoptotic cells treated with or without 5 mM glutamate for 12 hours in the presence or absence of 5 µM rottlerin. (ii) HT22 cells were transfected with either control or PKC siRNA (siPKC) and, after 24 hours, cells were exposed to 5 mM glutamate for 9 hours. Downregulation of PKC after transfection was confirmed by western blot analysis (right panel), and cell viability was measured by MTT assay (left panel). Each bar is a mean ± s.d. value from three separate experiments. ^*P<0.05, ^**P<0.01.

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