Itga5

Changes in the expression of the following proteins, such as ITGA5, FAK, and AKT, were detected by Western blotting. Proteins were lysed using RIPA lysis buffer (Thermo Fisher Scientific) and protease/phosphatase inhibitor (Beyotime Biotechnology), and their concentrations were detected by BCA (Thermo Fisher Scientific). Proteins were separated by 10% polyacrylamide gel electrophoresis, transferred to a PVDF membrane (GE Healthcare, Piscataway, NJ), and blocked with 5% skimmed milk powder for 1 h at room temperature. The membrane was then incubated overnight with primary antibodies on a 4°C shaker. The antibodies and concentrations used were the following: ITGA5 (Proteintech, 1 : 1000 dilution), FAK (Cell Signaling Technology, 1 : 1000 dilution), p-FAK (Cell Signaling Technology, 1 : 2000 dilution), AKT (Cell Signaling Technology, 1 : 1000 dilution), p-AKT (Cell Signaling Technology, 1 : 1000 dilution), and GAPDH (Cell Signaling Technology, 1 : 10000 dilution). Next, the membrane was incubated with the secondary antibody (Cell Signaling Technology 1 : 5000 dilution) at room temperature for 1 hr. Finally, the membrane was treated with ECL developer (Pierce), and the bands were developed using a gel imager FluorChem (USA, ProteinSimple).

The colonic tissues were washed with ice-cold PBS and lysed with lysis buffer (20 mM Tris at pH 7.5, 1 mM PMSF, 0.1% Triton X-100, and 10 μg/ml aprotinin). The concentration of protein was determined using a BCA assay (Sangon Biotech), and 20 μg of protein per lane was added on an 8-12% SDS-polyacrylamide gel. The protein electrophoretically transferred to a nitrocellulose membrane (0.1-μM pore size). The proteins were detected using rabbit polyclonal antibodies against mouse Tgfb-1, Col1a1, Itga5, and Gapdh (Proteintech Group) as primary antibodies and peroxidase-conjugated anti-rabbit IgG (Proteintech Group) as a secondary antibody. Protein was detected by an enhanced chemiluminescence system (ECL) and exposure to X-ray film.