Protease inhibitor cocktail (Roche Applied Science, Mannheim, Germany) containing
50 mM Tris-HCl, 150 mM NaCl, 1% NP-40 and 0.1% sodium dodecyl sulfate (SDS) was
added to the radioimmunoprecipitation assay buffer (pH = 8.0, MedChemExpress).
The protein was extracted by the addition of mixed solution to the cells. The
bicinchoninic acid protein quantification kit (Sigma-Aldrich Chemical Company)
was applied for protein quantification. Proteins were then subjected to 10%
SDS-polyacrylamide gel electrophoresis and transferred to a 0.45 µm
polyvinylidene fluoride membrane (Millipore Corporation, Billerica, MA, USA).
The membrane was sealed for 60 min with 5% bovine serum albumin and probed
overnight with primary antibodies to Cyclin D1 (sc-8396, Santa Cruz
Biotechnology Inc., Santa Cruz, CA, USA), β-catenin (ab22656, Abcam, Cambridge,
UK) and β-actin (sc-81178, Santa Cruz Biotechnology) at 4°C. The secondary goat
anti-mouse antibody (ab205719, Abcam) was used for a 1-h incubation at 25°C. The
immune response was detected using Super Signal West Femto Maximum Sensitivity
Substrate Kit (Thermo Fisher) and C-DiGit Blot Scanner (Gene Company, HK, China)
was used for image acquisition.
50 mM Tris-HCl, 150 mM NaCl, 1% NP-40 and 0.1% sodium dodecyl sulfate (SDS) was
added to the radioimmunoprecipitation assay buffer (pH = 8.0, MedChemExpress).
The protein was extracted by the addition of mixed solution to the cells. The
bicinchoninic acid protein quantification kit (Sigma-Aldrich Chemical Company)
was applied for protein quantification. Proteins were then subjected to 10%
SDS-polyacrylamide gel electrophoresis and transferred to a 0.45 µm
polyvinylidene fluoride membrane (Millipore Corporation, Billerica, MA, USA).
The membrane was sealed for 60 min with 5% bovine serum albumin and probed
overnight with primary antibodies to Cyclin D1 (sc-8396, Santa Cruz
Biotechnology Inc., Santa Cruz, CA, USA), β-catenin (ab22656, Abcam, Cambridge,
UK) and β-actin (sc-81178, Santa Cruz Biotechnology) at 4°C. The secondary goat
anti-mouse antibody (ab205719, Abcam) was used for a 1-h incubation at 25°C. The
immune response was detected using Super Signal West Femto Maximum Sensitivity
Substrate Kit (Thermo Fisher) and C-DiGit Blot Scanner (Gene Company, HK, China)
was used for image acquisition.