An371

Cells were harvested and lysed with RIPA buffer (Beyotime, Jiangsu, China) followed by BCA analysis to determine the protein concentration. The samples were separated by SDS-PAGE and transferred onto nitrocellulose membranes. After incubation with the primary antibodies anti-p65 (1:1000; AF0246, Beyotime, Jiangsu, China), anti-phosphor p65 (1:1000; AN371, Beyotime, Jiangsu, China), anti-GAPDH (1:10000; 10494–1-AP, Proteintech Group, Illinois, USA), anti-LAMB1 (1:5000; 23498–1-AP, Proteintech Group, Illinois, USA), the membranes were then incubated with a secondary antibody (1:5000; #7074, Cell Signaling Technology, Massachusetts, USA). After washing, signals were detected using a chemiluminescence system (Bio-Rad, California, USA) and analyzed using Image Lab Software (Bio-Rad, California, USA).

Caco-2 cells (purchased from China National Collection of Authenticated Cell Cultures, SCSP-5027) were a gift obtained from Professor Xian-qiong Zou, School of Biotechnology, Guilin Medical University. Sinapic acid (SA) was obtained from Aladdin Biochemical Technology Co., Ltd. (Shanghai, China). Nonessential amino acid (NEAA) solution and LPS were obtained from Solarbio Life Sciences Co., Ltd. (Beijing, China). TRNzol-A+ reagent, FastQuant RT Kit, and SuperReal PreMix Plus (SYBR Green) reagent were obtained from Tiangen Biotech Co., Ltd. (Beijing, China). Triton X-100, fix solution (4% paraformaldehyde), bovine serum albumin (BSA), goat serum (C0265), and other reagents were obtained from Beyotime Biotechnology Co., Ltd. (Shanghai, China). Primary antibodies against ZO-1 (AF8394), occludin (AF7644), claudin-1 (AF6504), NF-κB (AF7569), phospho-NF-κBp65 (AN371), phospho-IκBα (Ser32) (AF5851), phospho-IKKα/β (Ser176/180) (AI139), MyD88 (AF2116), TLR4 (AF8187), and β-actin (AF0003) were purchased from Beyotime Biotechnology Co., Ltd.