β actin

Western blot was performed as described previously [34 (link)]. A total of 40μg of protein lysates were separated by sodium dodecyl sulphate/polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred onto a polyvinylidene difluoride (PVDF) membrane (Bio-Rad, Hercules, CA, USA). The primary antibodies were against: DKK2 (ab38594, Abcam), active β-catenin (#05-665; Merck Millipore, Billerica, MD, USA), total β-catenin (#2677; Cell Signaling Technology, Danvers, MA, USA), c-Myc (#1472-1; Epitomics, Cambridge, MA, USA), cyclin D1 (#1677-1; Epitomics), occludin (ab31721; Abcam), vimentin (#2707-1; Epitomics), Ecad (#1702-1; Epitomics), N-cadherin (ab98952;Abcam), and β-actin (LK-ab008-100; Liankebio, China), and GAPDH (#AE082046; Beijing Biosynthesis Biotechnology, Beijing, China) was used as a control. Proteins were visualized using an enhanced chemiluminescence kit (Amersham Pharmacia Biotech, Piscataway, NJ, USA).

Cells were lysed with RIPA lysis buffer (Beyotime, China) mixed with protease inhibitors (Beyotime) under ice bath conditions for 30 min. Protein concentration was determined using the BCA Protein Quantification Kit (Beyotime). Proteins (50 μg) were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore, Bedford, MA, USA). The membranes were blocked in 5% nonfat milk for 2 h at room temperature and incubated with primary antibodies overnight at 4֩C. The membranes were incubated with horseradish peroxidase-labeled secondary antibodies (1:2000; Lianke bio, Hangzhou, China) for 2 h at room temperature, and β-actin was used as a protein loading control. Finally, an electrochemiluminescence detection system was used for visualization. The source of the antibodies and the concentrations used were rabbit anti-ADAM12 (1:1000, Abclonal, China) and β-actin (1:2000, Abclonal).