Activated β catenin

We extracted total protein using a radioimmunoprecipitation buffer kit (Sigma-Aldrich) and determined the protein concentration using a BCA protein assay kit (Beyotime, Shanghai, China). The proteins were separated by 8–12% sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to a polyvinylidene fluoride membrane. Next, the membranes were blocked in 10% defatted milk for 2–3 h and incubated for at least 12 h at 4°C with GAPDH (1:1000; No. 5174, Cell Signaling Technology, Shanghai, China), MLPH (1:500; No. 10338-1-AP, Proteintech, Wuhan, China), N-cadherin (1:1000; No. ab76057, Abcam), total β-catenin (1:1000; No. 848, Cell Signaling Technology), E-cadherin (1:1000; No. ab76319, Abcam), and activated β-catenin (1:1000; No. 19807, Cell Signaling Technology) antibodies. The secondary antibody horseradish peroxidase-conjugated IgG (1:8000, Proteintech) was then applied for 1 h at 37°C. GAPDH was used as the internal control for all WB assays. The internal control for nuclear protein was Histone H3. Bio-Rad Quantity One software (Bio-Rad, Hercules, CA, USA) was used to analyze protein expression.

Total protein was extracted using radioimmunoprecipitation assay (RIPA) buffer (Sigma-Aldrich, St. Louis, MO, USA), and protein concentrations were assessed using the BCA Protein Assay Kit (Beyotime, Shanghai, China). The total proteins separated via gel electrophoresis using a 10% sodium dodecyl sulphate polyacrylamide gel were then transferred to polyvinylidene fluoride membranes, which, after an approximately 60-min blocking in 5% bovine serum albumin, were incubated overnight at 4°C with β-actin (1:2000; Cell Signaling Technology, Danvers, MA, USA), NR1D2 (1:1000; R&D Systems), E-cadherin (1:1000; Abcam), N-cadherin (1:1000; Abcam), vimentin, activated β-catenin (1:1000; Cell Signaling Technology), and total β-catenin (1:1000; Cell Signaling Technology) antibodies. A horseradish peroxidase-conjugated goat anti-rabbit IgG (1:1000, Beyotime, Shanghai, China) secondary antibody was then applied for 60 min at 37°C. β-actin internally controlled all Western blots. The Bio-Rad Quantity One software (Bio-Rad, Hercules, CA, USA) helped quantify protein expression.