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Fig. 4 | Journal of Neuroinflammation

Fig. 4

From: Metabotropic glutamate receptor 5 inhibits α-synuclein-induced microglia inflammation to protect from neurotoxicity in Parkinson’s disease

Fig. 4

α-syn regulated mGluR5 degradation by lysosomes. a BV2 cells were transfected with myc-α-syn or vector for 48 h. mGluR5 expression in the cytoplasmic fraction was evaluated by western blotting, and quantification of mGluR5 level was normalized to β-actin (left). mGluR5 expression in the membrane fraction was evaluated by western blotting, and quantification of mGluR5 level was normalized to Na+-K+-ATPase (right). Cells transfected with vector served as control. **p < 0.01 versus control. b Western blotting analysis of mGluR5 levels under CHX (2 μg/mL) exposure for 0–12 h for the absence or presence of α-syn in BV2 cells. Quantification of mGluR5 levels was normalized to β-tubulin. Cells transfected with vector without CHX treatment served as control. *p < 0.05, **p < 0.01, and ***p < 0.001 versus CHX group. c, d BV2 cells were transfected with myc-α-syn or vector for 24 h followed by treatment with NH4Cl (1, 5, 10 mM) (c) or MG132 (1, 5, 10 μM) (d) for 12 h, and mGluR5 expression was detected by western blotting. Quantification of mGluR5 levels was normalized to GAPDH (c) and β-tubulin (d). Cells transfected with vector without drugs administration served as control. ***p < 0.001 versus control; #p < 0.05 versus α-syn transfection. e BV2 cells were transfected with myc-α-syn or vector for 24 h followed by treatment with NH4Cl (10 mM) or MG132 (10 μM), accompanied by CHX (2 μg/mL) treatment for 6 h or 12 h, and mGluR5 expression was detected by western blotting (top). Quantification of mGluR5 level was normalized to β-tubulin (bottom). Cells transfected with vector without drugs treatment served as control. ***p < 0.001 versus control, ###p < 0.001 versus CHX treatment for 12 h, &&p < 0.01 versus α-syn transfection with CHX treatment for 12 h. f Immunofluorescent co-stainings with anti-mGluR5 (red), anti-α-syn (green), and anti-LAMP-1 (blue) were conducted in primary microglia infected with LV-NC, LV-α-syn, or LV-α-syn with NH4Cl treatment. Arrowheads indicate examples of co-localization. Scale bar, 10 μm. g BV2 cells were transfected with myc-α-syn or vector for 24 h followed by treatment with NH4Cl (10 mM) or NH4Cl combined with MTEP (100 μM) for 12 h. Immunoblotting analysis of membrane fraction with an anti-mGluR5 antibody was conducted, and cell lysates with various antibodies were also detected (top). Quantification of protein bands was normalized to Na+-K+-ATPase (mGluR5), β-tubulin (COX-2, TNF-α, IL-1β), and β-actin (α-syn). The p-p38 and p-NF-κB p65 levels were normalized to total p38 and NF-κB p65, respectively (bottom). Cells transfected with vector without drugs administration served as control. **p < 0.01, ***p < 0.001 versus control; #p < 0.05, ##p < 0.01, and ###p < 0.001 versus α-syn transfection; &p < 0.05 versus α-syn transfection with MTEP treatment. Ctr, control; α-syn, α-synuclein. Data shown in this figure represent mean ± SEM (n ≥ 3). The statistical significance was determined by Student’s t test (a), two-way ANOVA (b), and one-way ANOVA followed by Dunnett’s test (ce, g). ns, not significant

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