Western Blot Analysis of Cell Signaling Proteins

The cells were lysed by cold RIPA buffer (Beyotime, Shanghai, China) for 30 min on ice, added to 5× SDS-PAGE loading buffer (Beyotime), and heated at 100 °C for 10 min. The lysates were then separated by 8–12% SDS-PAGE and transferred electrophoretically onto polyvinylidene difluoride (PVDF) membranes by Trans-Blot SD Cell and Systems (Bio-Rad) for 45 min at 15 V. After blocked with 5% non-fat milk in TBS-T buffer (20 mM Tris/HCl pH 8.0, 150 mM NaCl, 0.05% Tween 20), PVDF membranes were incubated with the primary antibodies [34 (link)]. After incubation with a secondary antibody, the signals were measured using a chemiluminescent imaging system (Tanon, Shanghai, China). Beta-ACTIN was used as an endogenous loading control. Antibodies used in this study were: Cyclin D1 (WL01435a, Wanleibio), CASPASE3 (WL02117, Wanleibio), β-Catenin (51067-2-AP, Proteintech, Chicago, IL, USA) AXIN1(16541-1-AP, Proteintech), AXIN2(20540-1-AP, Proteintech) and β-Actin (AC028, ABclonal, Wuhan, China).