Sappβ

Brain tissues were homogenized and sonicated in ice‐cold lysis buffer [50 mM Tris–HCl, pH 8.0, 140 mM NaCl; 1.5 mM MgCl₂; 0.5% NP‐40 with complete protease inhibitor cocktail (Roche)]. Protein concentration was measured in the supernatant by BCA Protein Assay (Dingguo). The monoclonal or polyclonal antibodies used were BACE1 (Abcam, 1:1000); APP full length (A8717, Sigma, 1:3000); sAPPα (6E10, Covance, 1:1000); sAPPβ (Covance, 1:500); ADAM10 (Abcam, 1:1000); PSEN1 (Proteintech, 1:500); GAPDH (Proteintech, 1:10000); tau46 (CST, 1:1000); phosphorylation‐tau 181 (CST, 1:1000); phosphorylation‐tau 202 (Abcam, 1:5000); and phosphorylation‐tau 396 (Abcam, 1:10000). The secondary antibodies were goat anti‐rabbit or anti‐mouse horseradish peroxidase‐labeled antibodies (Proteintech, 1:5000). The membranes were visualized using an ECL reagent (Thermo) and a Fusion FX5 image analysis system (VilberLourmat). Relative protein intensities were calculated using Quantity One software (Bio‐Rad).

Protein content was quantified by Bradford analysis prior to solubilization. 15 µg of protein plus LDS Sample buffer and 10% β-mercaptoethanol (Invitrogen NP0007) were separate by PAGE on a 4–12% Bis-Tris polyacrylamide gel (Biorad 3450125), transferred onto nitrocellulose using the Trans-blot Turbo system (Biorad) and visualized by red Ponceau staining. After membranes were blocked in 5%-milk (Biorad 1706404), the following primary antibodies were applied (overnight at 4°C, at 1:1000 dilution in blocking solution (Thermo 37573): Y188 (Abcam ab32136), 6E10 (BioLegend 803001), sAPPα (IBL 2B3), M3.2 (Biolegend 805701), sAPPβ (Covance Catalog# SIG-39138), sAPPβ-Sw (IBL 6A1), and GAPDH (Sigma G9545). After extensive washings in PBS/Tween20 0.05%, the following secondary antibodies were used diluted 1:1000 in 5%-milk: anti-mouse (Southern Biotech, 1031–05) and a 1:1 mix of anti-rabbit (Southern Biotech, 4050–05) and anti-rabbit (Cell Signaling, 7074). Secondary antibodies were incubated with membranes for 30 min, RT, with shaking). After extensive washings in PBS/Tween20 0.05%, blots were developed with West Dura ECL reagent (Thermo, PI34076), visualized with ChemiDoc MP Imaging System (Biorad) and signal intensities were quantified with Image Lab software (Biorad).