Rotiblock

Protein samples were boiled in a reducing sample buffer and separated in a SDS-Tris-glycine buffer through polyacrylamide gel electrophoresis (PAGE). Proteins were visualized by staining with Coomassie Brilliant Blue (Sigma-Aldrich Co.). For western blot, purified proteins were electrotransferred to Roti-PVDF membrane (Carl Roth GmbH & Co., Karlsruhe, Germany). The membrane was blocked with RotiBlock (Carl-Roth GmbH & Co.) blocking solution for 2 h at RT and rinsed in PBS with 0.1% Tween-20 (PBST). The membrane was then incubated for 1 h at RT with primary antibodies at working dilution in PBST with 10% RotiBlock and subsequently incubated with goat anti-mouse IgG (H+L)-HRP conjugate (Bio-Rad, Hercules, CA, USA) 1 : 4000 diluted in PBST with 10% RotiBlock. The enzymatic reaction was developed using 4-chloro-1-naphthol and H₂O₂ (Fluka, Milwaukee, WI, USA). For the analysis of MAb specificity, undiluted hybridoma supernatants were used. To check the purity of recombinant N protein, MAb against 6-His-tag epitope (Thermo Scientific, Rockford, IL, USA) was used as a primary antibody.

After treatment, the in vitro cultured astrocytes were harvested by using the lysis buffer (RIPA lysis buffer (complete), PMSF, Pepstatin, Sigma-Aldrich, Taufkirchen, Germany). The hippocampi of the slices were separated and homogenized in the ice-cold lysis buffer (RIPA lysis buffer (50× complete), 100× PMSF, Pepstatin, Sigma-Aldrich, Taufkirchen, Germany). The quantification of the proteins was determined by a bicinchoninic acid protein assay kit. For Western blotting, a total of 20 μL of the sample containing 30 μg protein was loaded per lane and the proteins were electrophoretically separated on an SDS-PAGE gel. The protein bands were transferred to a polyvinylidene fluoride membrane. After being blocked with Rotiblock (Biorad, South Granville, Australia) for 1 h, the membrane was incubated with Anti-MEGF10 antibody (1:500, Sigma-Aldrich, Taufkirchen, Germany, Cat No. ABC10) and anti-GAPDH (1:10,000, ThermoFisher, Kandel, Germany, Cat No. MA5-15738) 4 °C overnight. Expression of GAPDH was used as a loading control, and to assist in determining changes in MEGF10 protein expression. Subsequently incubated the membrane with horseradish peroxidase-conjugated secondary antibodies (1:10,000, CST, USA) for 2 h at room temperature. The bands were quantified by Imaging lab software.