Quantitative Western Blot Analysis

Western blot analysis was performed as described previously14 (link). Briefly, colon or cell proteins were subjected to SDS-PAGE and transferred to PVDF membranes, which were sliced for concurrent detection with different primary antibodies. β-actin (1:1000, catalog no. 66009-1-Ig, Proteintech, USA), Occludin (1:1000, catalog no. ab216327, Abcam, UK), ZO-1 (1:1000, catalog no. ab216880, Abcam, UK), PCNA (1:500, catalog no. AF0239, Affinity, USA), IL-1β (1:1000, catalog no. 12242S, CST, USA), IL-6 (1:1000, catalog no. DF6087, Affinity Biosciences, China), stat-3 (1:1000, catalog no. 12640S, CST, USA), p-stat-3 (S727) (1:1000, catalog no. ET1607-39, Huabio, China), non-phospho (Active) β-catenin (1:1000, catalog no. 8814S, CST, USA) and TNF-α (1:1000, catalog no. 41504, Signalway Antibody, USA). After incubation with the primary antibodies overnight at 4°C, the signals were detected, and protein band intensities were analyzed via densitometry in Image Lab and quantified in ImageJ software separately.