Tris buffered saline with tween 20

Proteins of jejunum were extracted using radioimmunoprecipitation assay buffer (Beyotime Biotechnology, Shanghai, China) containing phenylmethylsulfonyl fluoride (Beyotime Biotechnology, Shanghai, China), and total protein content was determined using a Bicinchoninic Acid Protein Assay Kit (Beyotime Biotechnology, Shanghai, China) according to the manufacturer’s protocol. Equal amounts of proteins (30 μg) were electrophoresed and then transferred on to polyvinylidene difluoride membranes (Millipore, Bedford, MA, USA). The membranes were incubated with the PCNA antibody overnight at 4 °C. Beta-actin was utilized as a loading control. After 6 washes in tris buffered saline with Tween-20 (Beyotime Biotechnology, Shanghai, China) for 5 min each, the membranes were incubated with secondary antibody for 60 min at room temperature. Finally, the blots were washed and the protein bands were detected using an enhanced chemiluminescence kit (Thermo Scientific, Wilmington, DE, USA). Signals were visualized using ChemiDoc Touch Imaging System (Bio-Rad Laboratories, CA, USA). The protein expressions were estimated by quantifying the intensities of the bands using Image J NIH software (Media Cybernetics, Rockville, MD, USA). The data of Western blotting were analyzed using 6 randomly selected samples per group (n = 6).

For biochemical analysis, the cells were washed with ice-cold phosphate-buffered saline (PBS; Beyotime Institute of Biotechnology, Jiangsu, China) and lysed in radioimmunoprecipitation assay lysis buffer [50 mM Tris, pH 7.4; 150 mM NaCl; 1% Triton X-100; 1% sodium deoxycho-late; 0.1% sodium dodecyl sulfate (SDS); Beyotime Institute of Biotechnology)]. The lysates were kept on ice for 30 min, followed by centrifugation at 12,000 × g for 25 min at 4°C. The clear lysate was then collected and β-catenin, axin, TCF4, E-cadherin, vimentin, NKD2, CXCR4 and β-actin proteins were separated by 12% SDS-polyacrylamide gel electrophoresis (30–50 µg protein/lane) and transferred to a polyvinylidine fluoride membrane (Beyotime Institute of Biotechnology). The membranes were incubated in 5% milk for 1.5 h, and then with β-catenin (1:1000), axin (1:1000), TCF4 (1:2000), E-cadherin (1:2000), vimentin (1:1000), NKD2 (1:1000), CXCR4 (1:1500) and β-actin (1:2000) antibodies diluted in non-fat milk overnight. The membranes were then washed with Tris-buffered saline with Tween-20 (Beyotime Institute of Biotechnology) and incubated with the HRP-conjugated secondary antibodies for 2 h. Immunoreactive proteins were visualized using a BeyoECL Plus kit (Beyotime Institute of Biotechnology).