Fast blocking solution

A RIPA lysis buffer (NCM, No.WB3100) and protease and phosphatase inhibitors were used to lyse rat hippocampal tissue. We measured the protein concentration of the extracts using the BCA protein analysis kit (Beyotime, No.P0010‐1). It was determined that an equal amount of protein (40 mg) was loaded onto the SDS–PAGE gel and then transferred to the PVDF membrane (Immobilon‐P Membrane, IPVH0010). Nonspecific protein binding was blocked with fast blocking solution (Beyotime, No. P0252‐100), followed by incubation with primary antibodies diluted in 1× TBS‐T for 8 h. Primary antibodies used include P‐ERK1/2 (diluted 1:5000, Proteintech, 28733‐1‐AP), ERK1/2 (diluted 1:1000, Proteintech, 11257‐1‐AP), P‐CREB (diluted 1:1000, Affinity, af3189), CREB (diluted 1:500, Abcam, ab32515), BDNF (diluted 1:5000, Abcam, ab108319), and β‐tubulin (diluted 1:5000, Proteintech, 10094‐1‐AP). After primary antibody incubation, the membrane was washed with 1× TBS‐T buffer and then incubated with HRP‐conjugated anti‐rabbit secondary antibody (diluted 1:5000, Proteintech, SA00001‐2) for 1 h. Following another wash, the membrane was visualized using an ECL chemiluminescence kit (NCM, P10100), and images were captured using the Tanon 5200 Multi imaging system (Tanon). Image data analysis was performed using ImageJ software (NIH).

Total proteins were extracted from intestinal tissues or cell lysates using RIPA buffer containing phosphatase inhibitors (Solarbio), and protein quantification was performed using the Kormas G-250 kit (Solarbio). Protein lysates were separated by 10% sodium dodecyl sulfate‒polyacrylamide gel electrophoresis (Beyotime, Beijing, China) and then transferred onto polyethylene fluoroethylene membranes (Millipore, Burlington, MA, USA). The membranes were blocked for 30 min at 25 °C with a blocking buffer consisting of 5 × fast blocking solution (Beyotime) in Tris-buffered saline and 0.1% Tween-20. The membranes were then incubated overnight at 4 °C with the primary antibody and then incubated with horseradish peroxidase-conjugated secondary antibody (Proteintech). Proteins were visualized using an electrochemiluminescence kit (Keygen Biotech, Nanjing, China) and a chemiluminescence imaging system (Bio-Rad, Hercules, CA, USA). The following primary antibodies were used: β-actin (1:2000 dilution, RRID = AB_2687938),anti-Occ (1:3000 dilution, RRID = AB_2880820), anti-CLDN1 (1:4000 dilution, RRID = AB_2079881), and anti-PAFH (1:3000 dilution, RRID = AB_2878146) (all from Proteintech). The experiments were performed in triplicate. The grey bands were calculated using ImageJ, and GraphPad 8.0 was used for ANOVA.