Western blotting assay
The total protein from Caco2 and IEC-6 cells and colon tissues were
homogenized using RIPA lysis buffer containing protease and 1 mM
phenylmethylsulfonylfluoride (PMSF). The protein concentrations were
tested using a BCA Protein Assay Kit (Beyotime Institute of
Biotechnology) according to the manufacturer’s protocol. The proteins
were separated by SDS-PAGE (7.5–15%), and then transferred to PVDF
membranes (Millipore, Massachusetts, USA). Finally, the membranes were
detected using enhanced chemiluminescence reagent with a Bio-Spectrum
Gel Imaging System, respectively (UVP, California, USA). Experiments
were performed at least five time.