Skimmed milk powder

The main reagents used in this study included TRIzol lysis buffer (Invitrogen, USA); reverse transcription kit: PrimeScript RT reagent kit with GDNA Eraser (RR 047A, Takara, Japan); qPCR kit: Tb Green Premix EX TAQ II (RR 820A, Takara); animal whole protein extraction kit (C510003, Sangon Biotech, China); BCA protein concentration assay kit (P0010, Beyotime Biotech, China); 5× loading buffer (P0015, Beyotime Biotech); SDS-PAGE gel preparation kit (P1200, Solar BioBeijing, China); SDS-PAGE electrophoresis solution (P00148, Beyotime Biotech); 10× EMT (D1060, Solar BioBeijing); methanol (CB/T693-1993, Shanghai Guangnuo Chemical Technology Co., Ltd., China); skimmed milk powder (D8340, Solar BioBeijing); 10×TBST buffer (powder) (T1087, Solar BioBeijing); nitrocellulose membrane (NC membrane) (37412133, PALL, USA); and ECL developer (34095, Thermo Fisher, USA).

Small intestinal tissues were homogenized in RIPA buffer (Solarbio, China) with a mixture of protease inhibitors. The Pierce BCA Protein Assay Kit (TransGen Biotech, China) was used to quantify protein concentration. Protein samples were subjected to electrophoresis using on 10% sodium dodecyl sulfate-polyacrylamide gels and transferred to PVDF membranes (MilliporeSigma, Burlington, MA, United States). Subsequently, the membranes were blocked with skimmed milk powder (Solarbio) and incubated overnight at 4°C with the following primary antibodies: anti-ZO-1 (ab96587, 1:1000 dilution; Abcam), anti-Occludin (A2601, 1:2000 dilution; ABclonal, Woburn, MA, United States), anti-Claudin-1 (ab307692, 1:1000 dilution; Abcam:1000), and anti-GAPDH (ab181602, 1:1000 dilution; Abcam). After five washes with TBST for 5 min each, the membranes were incubated with the respective secondary antibodies (goat anti-rabbit; SA00001-2, Proteintech, Rosemount, IL, United States) for 1 h at room temperature. Protein bands were visualized using a high-sensitivity ECL chemiluminescence kit (NCM Biotech, China) and gel imager (Bio-Rad Laboratories, Hercules, CA, USA), and band intensity was quantified using ImageJ software version 2.3.0 (National Institute of Health, Bethesda, MD, USA).