Sa00001 4

We extracted and denatured the total protein in each group of cells and tissues. After SDS-gelelectrophoresis, we transferred them to nitrocellulose membranes. Then, we incubated the different primary antibodies including TLR4 (1:500, ab13556, Abcam, UK), NLRP3 (1:1000, ab263899, Abcam), IL-1β (1:1000, ab234437, Abcam), IL-18 (1:4000, 60,070-1-Ig, Proteintech, USA), caspase-1 (1:1000, 22,915-1-AP, Proteintech), NF-κB (1:2000, 10,745-1-AP, Proteintech), ASC (1ug/mL, ab175449, Abcam), Pro-MMP9 (2ug/mL, MAB9111, R&D), Claudin5 (1:4000, ab131259, Abcam) and β-actin(1:5000, 66,009-1-Ig, Proteintech)overnight 4 °C.After washing the membranes three times, the blots were incubated with fluorescent-basedanti-rabbit(1:6000, SA00001-2, Proteintech), anti-mouse(1:5000, SA00001-1, Proteintech) or anti-goat(1:5000, SA00001-4, Proteintech) IgG secondary antibody at room temperature.We visualized the proteins by chemiluminescence detection reagent (Thermo Fisher, Waltham, MA, USA). We used Image J software to analyze the gray value of all bands quantitatively.

Total protein was extracted from the colon tissue samples via radio immunoprecipitation assay (RIPA) lysis buffer (WB-0071; Beijing Dingguo Changsheng Biotechnology Co., Ltd., Beijing, China). Protein samples were mixed with sample buffer (5X) and denatured by boiling. For immunoblots, proteins were separated by sodium dodecyl sulphate polyacrylamide gel electrophoresis on a 10% polyacrylamide gel. Samples were electrophoresed at 80–100 V for 2 h until the dye migrated to the bottom of the gel. The proteins were transferred to polyvinylidene difluoride membranes and the membranes were blocked in blocking buffer (5% non-fat dry milk) for 2 h at room temperature, and incubated with primary antibodies against CRF-R1 (ab59023; 1:500; Abcam, Cambridge, MA, USA) or p-p65 NF-_ΚB (3031; 1:1,000; Cell Signaling Technology, Inc., Danvers, MA, USA) and β-actin (wl01845; 1:1,000; Wanlei Biotechnology, Co., Ltd., Shenyang, China) overnight at 4°C. Membranes were then incubated with a horseradish peroxidase (HRP)-conjugated anti-goat (SA00001-4; 1:7,000; Proteintech Group, Inc., Rosemont, IL, USA) and anti-rabbit (Wla023a; 1:8,000; Wanlei Biotechnology, Co., Ltd.) secondary antibody for 1 h at room temperature. The proteins were visualized with a chemiluminescent substrate (WBULS0100; EMD Millipore, Billerica, MA, USA) using an imaging system (Bio-Rad Laboratories Inc., Hercules, CA, USA).