Intestinal Protein Expression Analysis

Western blotting was performed as previously described [25 (link)]. Lysis buffer with added protease and phosphatase inhibitors were used for extracting total protein from intestinal tissues in each group (Beijing Solarbio Science and Technology Co., Ltd., Beijing, China). After grinding tissue, the supernatant protein was collected and quantified via the bicinchoninic acid method. PVDF membranes were incubated with specific primary antibodies, which included rabbit monoclonals against occludin (1:1000; Abclonal), claudin-1 (1:1000; Abclonal), COX-2 (1:1000; Abcam), caspase-3 (1:1000; CST), Bcl2 (1:1000; Abclonal), Bax (1:1000; Abclonal), HMGB1 (1:1000; NOVUS), Nrf2 (1:1000; NOVUS), HO-1 (1:1000; Abclonal), and mouse polyclonals against GAPDH (1:1000; Abcam) and β-actin (1:1000; Abcam) at 4 °C overnight. After incubation with the primaries, the membrane was washed three times, then incubated with goat anti-rabbit (1:5000; Zhongshan Golden Bridge, China) and goat anti-mouse (1:5000; Zhongshan Golden Bridge, China) IgG antibodies at room temperature for 1 h, and then washed three times. Lastly, the membranes incubated with enhanced chemiluminescence were exposed to the ChemiDoc Touch Imaging System. The grayscale values of protein bands were analyzed using Image J.