Ab2721

Western blot analysis was performed, as previously described (17 (link)). Total lysis of the cells was conducted with RIPA buffers (Thermo Fisher Scientific, Inc.) and protein concentration was determined with a bicichoninic acid protein assay kit (Thermo Fisher Scientific, Inc.). Equal quantities (50 µg) of proteins were separated and transferred onto polyvinylidine difluoride membranes. The membranes were blocked with 5% non-fat dried milk, following which the membranes were probed overnight at 4°C with the following antibodies: Rabbit monoclonal anti-Wnt3a (Ab2721 1:1,000), rabbit monoclonal anti-p-GSK-3β (Ab5558; 1:2,000), rabbit monoclonal anti-p-β-catenin (Ab9561; 1:2,000), rabbit monoclonal anti-GSK-3β (Ab12456; 1:2,000), rabbit monoclonal anti-β-catenin (Ab4176; 1:2,000) or mouse monoclonal anti-β-actin (Ab3700; 1:2,000) (all from Abcam, Cambridge, MA, USA). This was followed by incubation with either horseradish peroxidase-conjugated goat anti-rabbit (ZB-2301) or anti-mouse antibody(ZB-2305) (1:5,000; Zhongshan Golden Bridge Biotechnology, Beijing, China) for 2 h at room temperature. Immunoreactive proteins were visualized using enhanced chemiluminescence, and signal intensity was detected and quantified using Alpha Imager (Alpha Innotech Corporation, San Leandro, CA, USA).