Bca assay method

Tissue samples (100 mg) were homogenized with Triton-HEPES buffer (Sigma Aldrich, St. Louis, MO). Total protein was determined using a bicinchoninic acid (BCA) assay method (Beyotime, Shanghai, China). Diaphragmatic cytokines including TNF-α, IL-1β, and IL-6 and systemic IL-6 were measured by commercial ELISA kits according to the manufacturer's protocols.

The cell pellets were lysed in an ice-cold mixture of RIPA buffer with PMSF (Beyotime, Shanghai, China), and total protein was extracted from the lysed cells after centrifugation in a refrigerated centrifuge. The protein concentration was measured using the BCA assay method (Beyotime). After denaturation in loading buffer at 100°C for 10 min, equal amounts of protein were loaded onto 8–20% SDS-PAGE gels for electrophoresis and electrophoretically transferred onto methanol-activated polyvinylidene difluoride (PVDF) membranes (0.45 mm; Millipore). The membranes were blocked with 5% BSA dissolved in TBST (20 mM Tris, 145 mM NaCl (pH 7.6) and 0.02% Tween 20) for 2 h at room temperature and then incubated with primary antibodies at 4°C overnight. The following day, the membranes were washed with TBST (three times for 15 min) and incubated with proper secondary HRP-conjugated antibodies for 2 h at room temperature. The immunoreactive bands were detected by reacting them with enhanced chemiluminescence (ECL) reagents (Pierce) under a ChemiDoc MP Imaging System (Bio-Rad).

Mucosal tissue samples from the jejunum and ileum were homogenized in ice‐cold PBS and centrifuged at 6000 × g for 20 minutes at 4°C, and the supernatant was used for enzyme analysis. The activities of intestinal alkaline phosphatase (ALP), lactase, sucrase, and maltase were analysed using commercially available swine ELISA kits according to the manufacturer's instructions (Jiangsu Yutong Biological Technology Co., Ltd.). The protein contents of WCLs were measured using the BCA assay method (Beyotime Institute of Biotechnology). The intestinal digestive enzyme activity was normalized to the protein concentration (mU/mg) of WCLs.