Ab183612

Rat cerebral tissues or cell lysates of the above groups were placed in ice-precooled RIPA lysis buffer (Beyotime) for 30 min, followed by centrifugation (12000 g, 10 min, 4° C). The supernatant was used for analysis. Protein concentration was determined using the BCA (Beyotime) method. Proteins were subjected to 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Beyotime) for separation, transferred to the nitrocellulose membranes (Whatman Inc., Piscataway, NJ, USA), and then blocked for 1 h with blocking solution (P0023B, Beyotime) at room temperature. Afterwards, the membranes underwent an overnight incubation with primary antibodies at 4° C, followed by membrane washing and then a 2-h incubation at room temperature with secondary antibody horseradish peroxidase-labeled immunoglobulin G (IgG) H&L (ab205718, 1:5000, ABCAm). The enhanced chemiluminescence working solution (EMD Millipore Corporation, Billerica, MA, USA) was used for development. ImageJ 1.48 (National Institutes of Health) was used for data analysis with β-actin as an internal reference. The primary antibodies (all from ABCAm) were neprilysin (NEP) (ab216341, 1:1000), insulin-degrading enzyme (IDE) (ab133561, 1:1000), BACE1 (ab183612, 1:1000), Wnt3a (ab219412, 1:1000), β-catenin (ab32572, 1:5000) and β-actin (ab8227, 1:1000).

The extracted proteins were separated by electrophoresis with 10% SDS-PAGE. Gel was run at 80 V for 20 min and 120 V for 60 min and then transferred onto PVDF membranes at 4°C at 80 V for 1.5 h. The target proteins APP, BACE1, and PKA were measured by incubating with the primary antibodies against APP (1 : 2000, MAB348, Millipore, USA), BACE1 (1 : 2000, ab183612, Abcam, USA), p-PKA (1 : 2000, ab32390, Abcam, USA), and PKA (1 : 2000, ab75993, Abcam, USA) at 4°C overnight. After washing three times with TBST, the corresponding secondary antibody was used at a dilution of 1 : 2000 (bs-40295G, bs40296G, Bioss, China), followed by visualization with the ECL kit (mixed with 1 : 1, PE0010, Solarbio, China). The exposure was completed in a dark room with the chemiluminescence gel imaging system (C600, Azure Biosystems, USA). The antibodies against GAPDH (1 : 2000, TA-08, Zsbio, China) and β-actin (1 : 2000, bs-0061R, Bioss, China) were used as internal controls. Quantitative results were expressed as a ratio of APP to GAPDH, BACE1 to β-actin, and p-PKA to PKA and then compared in each group to measure relative changes.

Cultured cells or brain tissues were homogenized in ice‐cold RIPA Lysis and Extraction Buffer (89901; Thermo, CA, USA) supplemented with a protease inhibitor cocktail (78439; Thermo, CA, USA). The protein concentration was determined using a BCA assay kit (23250; Thermos, CA, USA). Protein samples were separated by 8%–12% SDS–PAGE, blotted onto a polyvinylidene fluoride membrane (Millipore), blocked for 60 minutes in 5% milk and incubated overnight at 4℃ with rabbit monoclonal anti‐MOAB‐2 (1:2,000; ab126649; Abcam), mouse monoclonal anti‐β‐actin (1:4,000; A5441; Sigma), rabbit monoclonal anti‐BACE1 (1:1,000; ab183612; Abcam) and mouse monoclonal anti‐GAPDH (1:4,000; AF0006; Beyotime) antibodies. After three washes with TBST, the membranes were incubated with horseradish peroxidase‐conjugated goat anti‐mouse or goat anti‐rabbit secondary antibodies at room temperature for 2 hours. The immunoreactive bands were detected using an enhanced chemiluminescence reagent (ECL, Pierce) and quantified using ImageJ software.