Phosphatase cocktail and protease inhibitor cocktail

For Western blot analysis, cells were lysed in RIPA buffer (150 mM NaCl, 1% Triton X-100, 1% deoxycholate, 0.1% SDS, 10 mM Tris-HCl pH 7.2, 5 mM EDTA), containing the appropriate concentration of Phosphatase Cocktail and Protease Inhibitor Cocktail (Sigma-Aldrich). Protein concentration was measured by the BCA Protein Assay Kit (Termo Scientific Pierce, Rockford, IL, USA). The proteins were electrophoresed and blotted on Immobilon-P PVDF membranes (Millipore Co., MA, USA). Membranes were blocked in PBS-tween 0.1% with 5% non-fat dried milk for 1 h at 25 °C and then incubated with the first antibody overnight at 4 °C. After washing with PBS-tween 0.1%, membranes were subjected to the peroxidase-conjugated secondary antibody and developed by chemiluminescence (ECL, Amersham Pharmacia Biotech, Little Chalfont, UK) employing the high definition system ChemiDocTR XRS+ (Bio-Rad Laboratories, Hercules, CA, USA). The bands corresponding to the different proteins were digitalized employing the Image Lab version 3.0.1 (Bio-Rad Laboratories). This assay was performed three times for each protein.

For Western blot analysis, cells were lysed in RIPA buffer (150 mM NaCl, 1% Triton X-100, 1% deoxycholate, 0.1% SDS, 10 mM Tris–HCl pH 7.2, 5 mM EDTA), containing the appropriate concentration of Phosphatase Cocktail and Protease Inhibitor Cocktail (Sigma-Aldrich). Protein concentration was measured by the BCA Protein Assay Kit (Termo Scientific Pierce, Rockford, IL, USA). The proteins were electrophoresed and blotted on Immobilon-P PVDF membranes (Millipore Co., MA, USA). Membranes were blocked in PBS-tween 0.1% with 5% non-fat dried milk for 1 h at 25 °C and then incubated with the first antibody overnight at 4 °C (E-cadherin, β-catenin (BD Transduction Laboratories), N-cadherin, Vimentin, GSK3β, GAPDH (Abcam) and NF-κβ (Cell Signaling)). After washing with PBS-tween 0.1%, membranes were subjected to the peroxidase-conjugated secondary antibody and developed by chemiluminescence (ECL, Amersham Pharmacia Biotech, Little Chalfont, UK) employing the high-definition system ChemiDocTR XRS + (Bio-Rad Laboratories, Hercules, CA, USA). The bands corresponding to the different proteins were digitalized employing the Image Lab version 3.0.1 (Bio-Rad Laboratories).