Protein Expression Analysis by Western Blotting

Cells were lysed using ice-cold RIPA lysis buffer (Beyotime) and the protein concentrations were quantified. Western blotting assay was performed as previously described (32 (link)). The PVDF membranes were incubated with rabbit anti-cyclin D1 (1:1,000; Proteintech, Wuhan, China), c-Myc (1:1,000; Cell Signaling, Boston, MA, USA), cyclin A2 (1:1,000; Cell Signaling), β-catenin (1:1,000; Cell Signaling), MMP2 (1:1,000; Proteintech), MMP7 (1:1,000; Cell Signaling), BTRC (1:1,000; Cell Signaling), FBXW7 (1:1,000; Proteintech), and α-tubulin (1:2,000; Proteintech) primary antibodies at 4°C for overnight. After conjugated with HRP goat anti-rabbit IgG antibody (1:5,000; Abcam, Cambridge, MA, USA), immunoreactive bands were visualized by the Super ECL prime (US Everbright Inc., Suzhou, China) under a western blotting detection instrument (Clinx Science Instruments, Shanghai, China).