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Figure 7


Fig. 7. Inhibition of PKC{delta} prevents ubiquitylation and degradation of MKP-1 in primary immature cortical neurons. (A) (i) Immature primary cortical neuron cultures prepared from day E13 mouse fetuses were seeded onto 24-well plates and grown for 24 hours. Cells were then incubated with or without 5 mM glutamate (Glu) in the presence or absence of 20 µM U0126 or 3 µM rottlerin (Rott) for 24 hours as indicated. Cell viability was analyzed by MTT assay. Results are mean ± s.d. from three separate experiments. **P<0.01 compared with control cells exposed to glutamate alone. (ii) Representative photomicrographs of apoptotic cells incubated with or without 5 mM glutamate in the presence or absence of 20 µM U0126 or 3 µM rottlerin for 24 hours, detected by TUNEL staining. (B) Cells were transfected with either control or PKC{delta} siRNA (siPKC{delta}) and, after 24 hours, cells were exposed to 5 mM glutamate for 16 hours. Downregulation of PKC{delta} was confirmed by quantitative RT-PCR (ii), and cell viability was measured by MTT assay (i). Each bar is mean ± s.d. value from three separate experiments. **P<0.01. (C) Cells were incubated with or without 5 mM glutamate in the presence or absence of 20 µM U0126 or 3 µM rottlerin for 12 hours. Cells were then harvested, lysed and phospho-(p)ERK1/2 or total ERK1/2 in equivalent amounts of total lysate protein (40 µg) were visualized by western blot analysis using specific antibodies against phospho-ERK1/2, ERK1/2 and actin. (D) Cells were incubated with or without 5 mM glutamate in the presence or absence of 5 µM rottlerin for 14 hours, followed by addition of 5 µM MG132 (MG) for 4 hours. Equal amounts of proteins in each cell extract were subjected to immunoprecipitation (IP) using 2 µg of anti-MKP-1 antibody. The immunoprecipitates were subjected to immunoblotting (IB) using antibody against ubiquitin (anti-Ub; 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 two independent experiments.