Western Blot Analysis of Brain Samples

For Western blot analysis, brains were homogenised (FastPrep FP120, Qbiogene, Illkirch, France) at 10% (weight/volume, w/v) in RIPA buffer (150 mM NaCl, 1% NP-40, 0.5% DOC, 0.1% SDS, 50 mM Tris-HCl pH 8.0) and a subset of samples from comparable time points were digested with proteinase K (PK) (20 μg/ml) (Roche, Mannheim, Germany) for 1 h at 37 °C. Digestion was stopped by addition of 10× sample buffer and boiling for 10 min. Samples were analyzed by SDS-page (AnykD, Biorad, Hercules, USA), transferred to nitrocellulose membranes (0.2 μm pore size, BioRad) at 400 mA for 1 h, blocked for 1 h at room temperature in 5% milk powder in TBST buffer and incubated overnight at 4 °C with anti-PrP antibody Pom1 [50 (link)], Iba1 (Wako), Actin (Millipore), and GLP-1R (Santa Cruz) [63 (link)]. After washing and incubation for 1 h at room temperature with an HRP-conjugated anti-mouse or anti-rabbit secondary antibody (1:10.000 in blocking buffer), signals were detected with ECL femto reagent (Thermo Scientific) and visualized and quantified with a BioRad ChemiDoc imaging station and Biorad VersaDoc.