Chemidoctr xrs

Protein extracts were obtained using RIPA buffer/Triton pH 7.4 (Bioworld) with inhibitors of phosphatases (PhosSTOP EASYpack, Roche) and proteases (cOmplete ULTRA tablets Mini EDTA-free EASYpack, Roche), following commercial brand indications. Protein concentration was determined by BCA Protein Assay Kit (Thermo Scientific Pierce). Subsequently, extracts were diluted in Laemmli buffer (Bio-Rad), heated for 5 min at 98 °C and electrophoresis was performed in acrylamide/bisacrylamide gels in denaturing conditions (SDS-PAGE), using a Miniprotean base, and Western Blotting by Transblot Turbo system (Bio-Rad) to mmobilon-P PVDF membrane (Bio-Rad). Membranes were incubated with blocking solution of skimmed milk 5% in TBS with 0.1% Tween20, for at least one hour at room temperature shaking. Afterwards, the membranes were incubated with primary antibodies overnight at 4 °C shaking (Suppl. Table 1), washed with TBS-T and incubated with a secondary antibody conjugated with peroxidase (Suppl. Table 1) for 2 h at room temperature. Quimioluminiscence revealing (kit ECL Plus, Amersham) was carried out using the high resolution system ChemiDocTR XRS+ (Bio-Rad). Bands were digitalized using Image Lab version 3.0.1 (Bio-Rad) software.

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).