Detection of β-catenin and c-Myc protein expression by western blot analysis

RIPA was used to lyse the cells and extract total protein. Protein concentration was quantified by BCA. Samples of 0, 1, 2, 3 and 4 µl were added to the wells, respectively, and the concentration was adjusted to 20 µg/µl. A total of 40 µg protein/lane were separated via 12% SDS-PAGE and transferred onto a PVDF membrane. The corresponding bands were selected according to the target protein. The separated proteins were blocked with 5% of skim milk powder at room temperature for 1.5 h. TBST was used to wash the membranes for 3 min. After washing, 2 ml of western primary antibody solution consisting of β-catenin (1:1,000), c-Myc (1:500) and GAPDH (1:1,000) was added and the membranes were stored at 4°C overnight. On the next day, the membranes were incubated with primary antibodies for 30 min. Following the primary incubation, the membranes were incubated with HRP-labeled secondary antibody (1:1,000) for 1 h at 37°C and were rinsed 3 times with PBS for 5 min each time. Protein bands were visualized in a dark room. ECL was carried out and the protein expression levels were determined. GAPDH was used as an internal reference for the analysis of the relative expression of each indicator. The results were analyzed by Image Lab™ software (Bio-Rad Laboratories, Inc.).