Ecl plus western blotting substrate

Furthermore, 100 mg of the sample was taken in an RNAse-free tube with 1 ml of protein lysis buffer (Biosharp, Anhui, China) on ice for 30 min, then centrifuged at 12,000 rpm and the supernatant was taken. After measuring the supernatant protein concentration by the BCA kit (Thermo Fisher, USA), then the protein lysate diluted the supernatant to the same concentration. The diluted sample was mixed with the loading buffer (Beyotime, Shanghai, China) at a ratio of 1:5 and denatured at 100°C for 10 min. Then, the proteins were separated by 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE), and transferred onto a nitrocellulose membrane (BIO-RAD, USA). After blocking with blocking buffer (Beyotime, Shanghai, China), the membranes were incubated with the corresponding primary antibody at 4°C for 12–16 h, followed by incubation with horseradish peroxidase (HRP)-conjugated secondary antibody for 1 h. Bands were detected using the ECL PlusTM Western Blotting Substrate (CliNX, Shanghai, China). Band intensity was quantified using ImageJ software (ImageJ 1.52, National Institutes of Health, USA). Primary antibodies for β-actin (1:10,000), zonula occludens-1 (ZO-1, 1:250), Occludin (1:1,000), and Claudin-1 (1:1,000) (Abcam, MA, USA) were used in this study.