Fig 1: Immunoblot analysis of mGlu2 receptor and NF?B p65 protein levels in the hippocampus. mGlu2 protein levels in the hippocampus of CRS mice treated with saline and unstressed controls (Ctrl) are shown in (A); mGlu2 protein levels in the hippocampus of CRS mice treated i.p. with saline, MF (3 mg/kg), LAC (30 mg/kg) or their combination are shown in (B), where the immunoblot is representative of three blots used for the analysis and contains two samples from Ctrl mice for comparative purposes; NF?B p65 protein levels in the hippocampus of Ctrl mice and CRS mice treated with saline or MF (3 mg/kg) are shown in (C). Data are means ± SEM (A), Student’s t test, *p < 0.05 vs. Ctrl, t(12) = 2.302 (n = 7) (B) (C) One-way ANOVA + Fisher’s LSD (B) *p < 0.05 vs. saline and LAC, F(3,25) = 2.904; p = 0.0546 (n = 4–8) (C) *p < 0.05 vs. all other values, F(2,11) = 31.74; p < 0.0001 (n = 4–5).
Fig 2: The mGluR2 ectodomain soluble protein (mGluR2-GST) neutralizes the infectivity of RABV in a dose-dependent manner.mGluR2-GST neutralized ERA-eGFP infection of HEK293 cells (A), SK cells (B), N2a cells (C), and mPN cells (D), and neutralized VSV?G-ERAG-eGFP infection of HEK293 cells (E) but failed to neutralize VSV-eGFP infection of HEK293 cells (F). A one-way ANOVA was used for the statistical analysis. *, p<0.05. **, p<0.01. ***, p<0.001.
Fig 3: RABV and mGluR2 are internalized into cells and transported together in early and late endosomes.N2a Cells were stained by using the Tyramide Signal Amplification immunofluorescent method. Absence of co-localization of tubulin (green), Rab7 (red), and Tomm20 (purple) served as a negative control for co-localization (A and B). Significant co-localization of the mitochondrial marker AIF (green) and Tomm20 (red) served as a positive control for co-localization (C and D). N2a cells infected with ERA-N-mCherry for 20 minutes at 37°C were used to perform immunofluorescence staining for RABV antigen (red), mGluR2 (green), Rab5 or Rab7 (purple), and the cell nuclei (blue). Co-localization of the RABV-mGluR2 complex with Rab5 (E, F) or Rab7 (H, I) was observed and counted. The images, comprising three single fluorescence channels (G, J), represent amplified random co-localization spots in the merged image within the small white box (G from E, F from J). The 3D-rendered images were generated by using Imaris software (G, J) and the co-localization of the RABV-mGluR2 complex with Rab5 (G) or Rab7 (J) from the three single fluorescence channels is indicated with the white arrowhead.
Fig 4: DHK blocks Cef-induced improvement on recognition memory deficits and suppression on the mGluR2 protein expression in APP/PS1 mice. The histograms of (A,B) show the recognition index in test 1 (A) and test 2 (B) of the novel object recognition test. (C) Shows the expression of mGluR2 assayed by the Western blot analysis. The upper is a representative immunoblot and the lower is the quantitative presentation of the immunoblot with IOD. The number of mice for each test is shown in Table 1. The italic values upon the bars are statistical P values.
Fig 5: RABV binding results in the downregulation of cell surface mGluR2.Flow cytometry detected cell surface mGluR2 on RABV ERA-infected HEK293 cells (ERA), which was compared to uninfected HEK293 cells by staining with anti-mGluR2 monoclonal antibody (mGluR2) or isotype antibody control (IgG2a) at 4°C or 37°C (A). The flow cytometry results were analyzed with a one-way ANOVA (B). *, p<0.05. **, p<0.01.
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