Claudin 2

Total proteins from jejunal epithelium were extracted through RIPA lysis buffer (#AR0108, BOSTER). The mitochondrial proteins were extracted using the Tissue Mitochondria Isolation Kit (Beyotime). A BCA Protein Assay Kit (Beyotime) quantified protein concentration following the manufacturer’s protocols. Certain amounts of proteins (~30 μg) were loaded onto each lane of SDS-PAGE gel to undergo electrophoresis, then transferred onto PVDF membranes (Millipore, Bedford, MA, USA). After blocking and washing, the membranes were incubated with specific primary antibodies (below) at 4°C overnight. Membrane incubation of corresponding HRP-conjugated secondary antibodies was conducted at room temperature for 1 h. Subsequently, ECL reagents (#E170-01, GenStar) were prepared and added to the membranes for chemiluminescence, which were imaged and captured using a ChemiDoc Touch imaging system (BIO-RAD, Hercules, CA, USA). Primary antibodies used in this study were: Occludin (#AF7644, Beyotime), Claudin 1 (#28674-1-AP, Proteintech), Claudin 2 (#AF0128, Affinity, Changzhou, China), IL-6 (#500286, ZEN BIO, Chengdu, China), Caspase-3 (#19677-1-AP, Proteintech), Caspase-9 (#10380-1-AP, Proteintech), AIF (#AA306, Beyotime), Calpain-1 (#381868, ZEN BIO), Cox-IV (#200147, ZEN BIO), and β-actin (#T0022, Affinity) (internal reference for total protein).

RIMVECs were seeded, divided into groups, and treated as described in the in vitro experiment paragraph. Total proteins were extracted using the radio immunoprecipitation assay (RIPA) cell lysis buffer for 10 min, and protein concentration was determined using the bicinchoninic acid (BCA) protein detection kit (Beyotime Biotechnology, China). A standard western blot protocol was performed, and the following primary antibodies were used overnight at 4°C: TLR4 (1:800) (ABclonal, Wuhan, China), NLRP3 (1:1000) (ABclonal), GSDMD (1:1000) (ABclonal), caspase-1 (1:1000) (ABclonal), ASC (1:1000) (ABclonal), NF-κB p65 (1:1000) (Abmart, Shanghai, China), p-NF-κB p65 (1:400) (Abmart), p-I-κB (1:1000) (Abmart), I-κB (1:1000) (Abmart), IL-18 (1:1000) (Affinity Biosciences, Colorado, USA), IL-1β (1:1000) (Affinity Biosciences), claudin-1 (1:1000) (Affinity Biosciences), claudin-2 (1:1000) (Affinity Biosciences), ZO-1 (1:1000) (Affinity Biosciences), and β-tubulin (1:1000) (Affinity Biosciences) used as loading control, followed by the incubation with HRP (Horseradish Peroxidase)-conjugated secondary antibodies (1:5000) (ABclonal) for 1 h at room temperature. The blots were visualized using a Tanon 5200 chemiluminescence imaging system (Shanghai, China) and quantified using the ImageJ software (National Institutes of Mental Health, Bethesda, MD, USA).