Figure 4

Induction of endoplasmic reticulum (ER)-stress markers by blockage of mGluR5. (A) Immunoblot analyses to detect GRP78 protein were performed on lysates of BV-2 cells that had been treated with 100 μM MPEP for the indicated time period. β-actin served as a loading control. (B) Densitometric analysis of GRP78 band normalized against β-actin. (C) Cells were preincubated for 1 hour with 1 μM BAPTA-AM or 1 mM sodium phenylbutyrate (PBA), followed by further incubation with 100 μM MPEP for 24 hours. Expression of CHOP, GRP78 and GRP94 mRNA were measured by quantitative PCR. (D) Cells were pre-exposed for 1 hour with 1 mM AICAR or 1 μM compound C, followed by a further incubation with 100 μM MPEP for 2 hours and GRP78 protein levels were analyzed by immunoblotting. (E) Densitometric analysis of GRP78 band normalized against β-actin. (F) Cells were pre-exposed for 1 hour with 1 mM AICAR or 1 μM compound C, followed by a further incubation with 100 μM MPEP. Expression of CHOP, GRP78 and GRP94 mRNA were measured by quantitative PCR and mRNA expression levels were normalized to GAPDH control. Results represent mean ± SD of three independent experiments. Significance was analyzed by one-way ANOVA followed by Tukey’s tests. **P <0.01; ***P <0.005. CHOP, C/EBP homologous protein; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; MPEP, 2-methyl-6-(phenylethynyl)-pyridine.