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Fig. 1. ERK1/2 contributes to glutamate-induced cell death in HT22 cells. (A) HT22 cells were incubated with 5 mM glutamate (Glu) for the indicated times. Cell extracts were subjected to SDS-PAGE and immunoblotted with antibodies specific for phospho-ERK1/2 (pERK), phospho-JNK (pJNK), or phospho-p38 (pp38) MAPK. Membranes were stripped and reprobed for total ERK1/2, JNK or p38 as a control. Shown are blots from a representative experiment out of four separate experiments. (B) HT22 cells were incubated with 5 mM glutamate for 12 hours in the presence or absence of 20 µM U0126 (Glu/U0126), 10 µM SB202190 (Glu/SB) or 20 µM SP600125 (Glu/SP). Shown are representative light microscopic images. Results from the MTT assay are also presented. Each bar is a mean ± s.d. value from four separate experiments. (C) HT22 cells were exposed to 5 mM glutamate for 7 hours in the presence or absence of 20 µM U0126. Phosphorylation of ERK1/2 in whole cell extracts was determined by immunoblotting with an antibody against phospho-ERK1/2. Blots shown are representative of four separate experiments. (D) Representative photomicrographs of apoptotic cells treated with or without 5 mM glutamate for 12 hours in the presence or absence of 20 µM U0126. (E) HT22 cells were incubated with 5 mM glutamate for the indicated times. Cell extracts were immunoblotted with an antibody specific for phospho-ERK1/2 or phospho-MEK1/2. Membranes were stripped and reprobed for actin as a control. Results were reproducible, and blots shown are representative of three separate experiments. (F) HT22 cells were exposed to 5 mM glutamate, and then 20 µM U0126 was added either simultaneously or at the indicated times after the addition of glutamate. Cell viability was analyzed after 12 hours by the MTT conversion assay. Results are mean ± s.d. value from four separate experiments.
