Protein Expression Analysis by Western Blot

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).