Chemidoc touch imaging system

Total protein was extracted from hippocampal tissues with protein lysis buffer (G2002, Servicebio, China). The protein concentration was determined with a BCA kit (E-BC-K318-M, Elabscience, China). The proteins in each sample were separated by 10% SDS–polyacrylamide gel electrophoresis and transferred onto polyvinylidene fluoride (PVDF) membranes. Western blot analysis was performed using the following primary antibodies raised against target proteins: rabbit anti-BDNF (1:2000, 66292-1-IG, Proteintech), rabbit anti-Erk1/2 (1:1000, T40071, Abmart), rabbit anti-phospho-Erk1 (T202/Y204) + Erk2 (T185/Y187) (T40072, Abmart), and rabbit anti-β-actin (1:2000, P30002, Abmart) antibodies overnight at 4 °C. Goat anti-rabbit horseradish peroxidase (HRP)-conjugated secondary antibody (1:10000 dilution, HY-P8001, MedChemExpress) was used. Western blotting substrates were visualized using ECL reagents with enhanced chemiluminescence detection and the Chemidoc™ Touch Imaging System with Image Lab software (Bio-Rad, Hercules, CA, USA). When several target proteins have similar molecular weights, we use the western blot fast stripping buffer (EpiZyme, Shanghai, China, PS107) for treatment and re-blocking. We declare and ensure that our western blot data comply with the digital image and integrity policies.

Cells were lysed in radioimmunoprecipitation assay (RIPA) buffer (89901; Thermo Fisher Scientifc) supplemented with protease inhibitors (04693132001; Roche, Basel, Switzerland) for 20 min on ice, followed by centrifugation at 13 000g for 20 min at 4°C to isolate the protein. Protein concentrations were determined using a bicinchoninic acid (BCA) assay (23225; Thermo Fisher Scientific). Equal amounts of protein were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) on 10% polyacrylamide gels and subsequently transferred onto polyvinylidene fluoride (PVDF) membranes (ISEQ00010; Millipore). Membranes were blocked in 5% non-fat milk in Tris-buffered saline with 0.1% Tween-20 (TBST; 0.1%) for 1 h at the room temperature. After washing three times with TBST for 10 min, anti-GFP mouse monoclonal antibodies (1:2000; AB1218; Abcam, Cambridge, UK) and glyceraldehyde-3-phosphate dehydrogenase-horseradish peroxidase conjugate (GAPDH-HRP; 1:2000; #3683; Cell Signaling Technology, Danvers, MA, USA) were incubated overnight at 4°C. After washing three times with TBST, the membranes were incubated with HRP-conjugated anti-mouse secondary antibodies (1:5000; #7076; Cell Signaling Technology) for 1 h at the room temperature. Protein bands were detected using ECL reagent (WBKLS0500; Millipore) and visualized on a ChemiDoc Touch imaging system (Bio-Rad, Hercules, CA, USA). Band intensities were quantified using ImageJ software (NIH, Bethesda, MD, USA), and protein expression levels were normalized to GAPDH. Results were analyzed from three independent experiments.