2.4. Western Blotting

Distal ileal tissues were lysed with radioimmunoprecipitation assay (RIPA) lysis buffer (Epizyme Biotech, Shanghai, China) with a 1% protease inhibitor (Epizyme Biotech, China). The homogenates were settled on ice for 1 h and centrifuged at 10,000× g for 10 min at 4 °C, and the supernatants were collected. The extracts were mixed with SDS loading buffer (Yeason, Shanghai, China), and then the solution was heated at 100 °C for 10 min. Ten microliters of the solution were loaded into a 10% SDS-PAGE gel produced by using a PAGE gel fast preparation kit (Epizyme Biotech, China) for electrophoresis, then transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, Burlington, MA, USA). The membranes were blocked with 3% bovine serum albumin (BSA) for 1 h at room temperature and incubated overnight at 4 °C with the antibodies (1:1000): claudin 1 (ABclonal, Shanghai, China); tubulin (Yeason, Shanghai, China). The next day, the membranes were washed with Tris Buffered Saline with Tween-20 (TBST) three times and incubated with peroxidase-conjugated goat anti-rabbit IgG(H1L) (Yeason, China) for 1 h at room temperature. The membranes were washed three times with TBST again. Protein bands were observed using horseradish peroxidase (HRP) substrate peroxide solution (Epizyme Biotech, China) and an Amersham 600 imager (General Electric, Burlington, MA, USA). The density of each band was measured by ImageJ v1.8.0. software.