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Fig. 6. PKC activation by glutamate induces ubiquitylation and downregulation of MKP-1 in HT22 cells. (A) HT22 cells were incubated with or without 5 µM ALLN or 5 µM lactacystine for 7 hours in the presence or absence of 5 mM glutamate (Glu). Western blot analysis was performed using specific antibodies against MKP-1 and actin. Relative MKP-1 protein levels were calculated by averaging the results obtained from three independent experiments. (B and C) Cells were treated with 5 mM glutamate in the presence or absence of 5 µM rottlerin (Rott) for 4 hours, followed by addition of 5 µM MG132 (MG) for 3 hours. Cells were then harvested, and subjected to immunoprecipitation (IP) of MKP-1 using anti-MKP-1 antibody. Following SDS-PAGE, membranes were immunoblotted (IB) with an anti-ubiquitin (anti-Ub) antibody (upper panel). The level of MKP-1 was determined by western blot analysis of the same cell lysates (lower panel). Results were reproducible, and blots shown are representative of three separate experiments.

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