Protein inhibitor

Ruminal epithelium and jejunal mucosal samples were homogenized in lysis buffer containing protein inhibitors (Beyotime Biotechnology, Shanghai, China). Total protein was extracted from tissues, and transferred to nitrocellulose membranes (Liu et al., 2013 (link)). Immunoblotting was conducted using rabbit anti-CLDN1 (1:2,000 dilution), rabbit anti-OCLN (1:2,000 dilution), and rabbit anti-ZO1 (1:2,000 dilution) antibodies (all from Proteintech). After several washes with Tris Buffered Saline Tween, membranes were incubated with a 1:5,000 dilution of goat anti-rabbit secondary antibody (Thermo Fisher Scientific, Shanghai, China) at room temperature for 2 h. The membranes were incubated with a 1:100,000 dilution of rabbit monoclonal β-actin antibody (Thermo Fisher Scientific, Shanghai, China) to normalize the results. Immunoreactivity was determined via chemiluminescence with ECL Plus WB substrate (Thermo Fisher Scientific, Shanghai, China) reference to the instructions and visualized by luminescence imaging (Tanon Technology, Shanghai, China). The density of the blotting band was analyzed by ImageJ software (version 1.46r, National Institutes of Health, USA). Results of each protein are presented as a ratio of densitometry to corresponding β-actin value.

Total protein was obtained from TFK1 and QBC939 cells using RIPA lysis buffer containing protein inhibitors (Beyotime), and total protein was assayed using a BCA kit (Beyotime). Subsequently, 20 µg of each sample was separated by SDS-PAGE and transferred onto polyvinylidene difluoride (PVDF) membranes (Millipore). The membranes were blocked with 5% bovine serum protein (BSA). After 1 h, membranes were incubated overnight at 4 °C with primary antibodies against Bax (#2772, CST), Bcl-2 (ab196495, Abcam), Wnt3a (26744-1-AP, Wuhan Sanying Biotechnology), β-catenin (#8480, CST), p-GSK3β (#5558, CST), GSK3β (#12,456, CST), c-Myc (ab32072, Abcam), Cyclin D1 (ab134175, Abcam), and GAPDH (ab181602, Abcam). The next day, membranes were washed five times with TBST and incubated with horseradish peroxidase-labeled secondary antibodies (AS1107, ASPEN). After 2 h, the bands were visualized using an ECL luminescent solution (Beyotime). However, during the western blot experiments, the corresponding membrane was firstly cut out according to the molecular weight of the target protein prior to hybridisation with antibody, and then incubated with the primary antibody. Thus, the original image was not a full length membrane.

Cells were washed twice with ice-cold PBS and lysed using 120–200 μl standard RIPA buffer containing protein inhibitors (Beyotime, Jiangsu, China). Protein in loading buffer was denatured by heating at 100°C for 10 minutes and then separated on a SDS-polyacrylamide gel by electrophoresis. 40–60 μg of protein per lane was loaded. The proteins were transferred onto a PVDF membrane (Bio-Rad, CA). The membranes were incubated in primary antibodies overnight at 4°C and then incubated in HRP-conjugated secondary antibodies for 2 hours at room temperature. The signals were visualized with ECL Plus substrates (Roche, Basel, Switzerland).