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Fig. 4. MKP-1 regulates ERK1/2 phosphorylation during glutamate-induced oxidative toxicity. (A) HT22 cells were incubated with 5 mM glutamate (Glu) for the indicated times. The cells were then extracted to determine protein levels of MKP-1 by western blot analysis. Western blot analyses with anti-actin antibody in the same extracts are shown in the bottom panels. The results are from a representative experiment out of four separate experiments. (B) Cells were transfected with 2 µg of Myc-tagged vectors containing either wild-type MKP-1 or its catalytically inactive mutant (MKP-1CS), expression was allowed for 12 hours, and then cells were exposed to 5 mM glutamate for 9 hours. Whole cell extracts were subjected to western blot analysis using specific antibodies against phospho-ERK1/2 (pERK1/2), ERK1/2 and MKP-1. (C) HT22 cells were transfected with scrambled control (Scr) or MKP-1 siRNA (siMKP-1 73). (i) The level of MKP-1 at 24 hours after transfection was monitored by western blot analysis. The levels of phospho-ERK1/2, ERK1/2, PKC
and GAPDH were also examined by western blot analysis. (ii) Phosphorylation of ERK1/2 under glutamate exposure for 7 hours was determined by western blot analysis. Relative phospho-ERK1/2 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) Cells were transfected with either control (scrambled, Scr) or MKP-1 siRNAs [siMKP-1 73 (si 73) and siMKP-1 853 (si 853)]. After 24 hours, cells were treated with 5 mM glutamate for 7-9 hours, followed by western blot analysis. *P<0.05, **P<0.01.
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