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Fig. 8. Role of MKP-1 in the regulation of glutamate-induced oxidative toxicity. (A) Exogenous expression of MKP-1 increases cell viability during oxidative toxicity. Cells were transfected with 2 µg of Myc-tagged vectors containing either wild-type MKP-1 or mock. After 12 hours, cells were exposed to 5 mM glutamate (Glu) for 9 hours. MKP-1 protein levels were determined by western blot analysis (right panel), and cell viability was measured by MTT assay (left panel). Results are mean ± s.d. obtained from three separate experiments. **P<0.01. (B) HT22 cells were transfected with control (Scrambled) or MKP-1 siRNA (siMKP-1 73) as indicated, expression was allowed for 24 hours, and then cells were exposed to 5 mM glutamate for 7 hours. Downregulation of MKP-1 after transfection was confirmed by western blot analysis (right panel) and cell viability was measured by MTT assay (left panel). Results are mean ± s.d. obtained from three separate experiments. (C) Immature primary cortical neuron cultures prepared from day E13 mouse fetuses were seeded onto 48-well plates and grown for 24 hours. (i) Cells were transfected with control or MKP-1 siRNA (siMKP-1 73; si 73) as indicated, expression was allowed for 24 hours, and then cells were exposed to 5 mM glutamate for 16 hours. Downregulation of MKP-1 after transfection was confirmed by western blot analysis (right panel), and cell viability was measured by MTT assay (left panel). (ii) Phosphorylation of ERK1/2 under glutamate exposure for 12 hours was determined by western blot analysis. Relative phospho-ERK1/2 (pERK1/2) levels were determined by densitometric scanning of enhanced chemiluminescence-exposed film, and the levels were calculated by averaging the results obtained from three independent experiments. (D) HT22 cells were transfected with either control or MKP-1 siRNA and, after 24 hours, cells were exposed to 5 mM glutamate in the presence or absence of 20 µM U0126 for 7 hours. Cell viability was measured by MTT assay. Results are mean ± s.d. obtained from three separate experiments. *P<0.05, **P<0.01.
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