Western Blot Analysis of Inflammatory and Tight Junction Proteins

Proteins were extracted from colon tissues with RIPA lysis buffer (Beyotime, Shanghai, China) containing phenylmethyl sulfonyl fluoride. A bicinchoninic acid protein assay kit (Beyotime) was used to measure the concentration of protein. Suitable quality protein samples were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to PVDF membranes (Millipore, Billerica, MA, USA). The PVDF membranes were blocked with 8% skim milk at room temperature for 1 h and incubated with primary antibodies at 4 °C overnight. The corresponding secondary antibodies were used at room temperature for 1 h, and the protein signals were detected with a FluorChem FC3 system (ProteinSimple, California, USA) using an enhanced chemilusystem reagent (Thermo Fisher, Waltham, USA) according to the manufacturer’s instructions. The antibodies used in this study were as follows: TNFα (1:200, Santa Cruz Biotechnology, Texas, USA), IL-1β (1:1000, Cell Signaling Technology, Massachusetts, USA), IL6 (1:200, Santa Cruz Biotechnology), ZO-1 (1:1000, Invitrogen, California, USA), Occludin (1:1000, Invitrogen), P62 (1:10,000, Abcam, Cambridge, USA), LC3 (1:1000, Proteintech, Chicago, America), CB1 (1:1000, Proteintech), GAPDH (Abclonal, Wuhan, China), horseradish peroxidase (HRP)-linked goat anti-rabbit IgG, and HRP-linked goat anti-mouse IgG (1:4000, Antgene, Wuhan, China).