Sample loading buffer

Total protein was extracted from tissue and cell samples with RIPA lysis buffer (Beyotime) supplemented with 1 mM phenyl methane sulfonyl fluoride, 4 µl/ml of cocktail, and phosphatase inhibitors. The protein concentration was measured using the BCA Protein Assay Kit (Thermo Fisher Scientific) according to manufacturer’s instructions. 20 µg protein was solubilized in sample loading buffer (Beyotime) and heated at 98°C for 12 min. The obtained proteins were separated by using SDS–PAGE electrophoresis and transferred to PVDF membrane (Millipore). Membranes were incubated with indicated primary antibodies at 4°C overnight and HRP-conjugated secondary antibodies at room temperature for 1 h. The chemiluminescence gel imaging system (Sagecreation) and high-sensitivity ECL Kit (Thermo Fisher Scientific) were used to detect the protein expression level. Cellular cytoplasmic and nucleic proteins were obtained by using nucleic and cytoplasmic protein extraction kit (Beyotime) according to the manufacturer’s instructions. The antibodies used in this study are listed in Table 3.

On completion of cell lysis using RIPA buffer (Beyotime Biotechnology, Shanghai, China), the obtained protein samples were detected to confirm protein concentration using BCA kit (Beyotime Biotechnology) and corresponding volume of them was taken to mix with sample loading buffer (Beyotime Biotechnology). Then, protein denaturation was achieved by 5 min heating in a boiling water bath. After protein sample preparation, the next step was to separate proteins by SDS-PAGE and to transfer them onto a PVDF membrane. On completion of 1 h blocking step with 5% skimmed milk at ambient temperature, primary antibodies GAPDH (5174S), MMP2 (40994S), E-cadherin (14472S), vimentin (5741SA), CD44 (37259S), OCT4 (2750S), SOX2 (3579S), BTG2 (ab197362, Abcam, Boston, USA) served to overnight co-incubation with the proteins at 4°C in a shaker. All primary antibodies were diluted as 1:1000, and except primary antibody BTG2, the others were provided by Cell Signaling, Boston, USA. The next day, after three rinsing steps (10 min/time), the proteins were incubated with secondary antibody (horseradish peroxidase (HRP)-labeled goat anti-rabbit IgG, 1:5000, CoWin Biosciences, Beijing, China) for 1 h at ambient temperature, followed by three washing steps again (10 min/time). After dropping the developer on the membrane, the detection was completed using a chemiluminescence imaging system (Bio-Rad).

Cells cultured in six-well plates were harvested with lysis buffer (Beyotime, China) and boiled for 10 min with sample loading buffer (Beyotime, China). The samples were then resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and proteins were transferred to polyvinylidene difluoride membranes (Millipore, USA). The membranes were blocked with 5% bovine serum albumin at room temperature for 2 h and incubated with rabbit polyclonal antibody against APN or mouse monoclonal antibody against PDCoV N or TGEV N for 3 h. After washing three times, the membranes were incubated with horseradish peroxidase-conjugated anti-polyclonal or -monoclonal antibody for another 1 h and washed three times. Proteins were detected using a western blot analysis system (Bio-Rad).