Emt sampler kit

Cellular pellets were collected and lysed using the radio-immunoprecipitation assay (RIPA) buffer (Sigma-Aldrich Corp., USA) supplemented with protease and phosphatase inhibitor cocktail and probed against primary antibodies including E-cadherin, Snail, Slug, N-cadherin, Zeb-1, vimentin, and β-catenin (antibodies included in EMT sampler kit, Cell Signaling Technology, Inc., Danvers, MA, USA); Nf-κB p65 (Cell Signaling Technology, Inc.); phospho-Akt (Ser 473) (Cell Signaling Technology, USA); Met, CD44, c-MYC (antibodies included in Wnt/β-catenin activated targets sampler kit, Cell Signaling Technology, Inc.); Klf4 (R&D Systems, Inc.); Nanog (R&D Systems, Inc.), GAPDH (Cell Signaling Technology, Inc.).

The samples were lysed in RIPA buffer (50 mM Tris, 150 mM NaCl, 0.1% SDS, 0.5% Sodium deoxycholate, 1% Igepal CA630, 2 mM EDTA, 50 mM NaF, pH 8) supplemented with a protease inhibitor cocktail (Roche Molecular Diagnostics, Pleasanton, California, CA, USA) and titrated using the DC Protein Assay (Bio-Rad Laboratories, Hercules, California, CA, USA). Amounts of 5 to 20 μg of proteins were separated by SDS-PAGE and transferred onto nitrocellulose membranes (Amersham, Little Chalfont, UK). The membranes were blocked 1 h at room temperature, incubated overnight at 4 °C with primary antibodies (EMT sampler kit, 9782 Cell signaling (Danvers, Massachusetts, MA, USA) and B-Actin, A2066 Sigma-Aldrich (Sigma-Aldrich Corp., St. Louis, MO, USA), incubated for 1 h with secondary antibodies (Bio-Rad Laboratories, Hercules, California, CA, USA), and developed using a standard ECL protocol. The quantifications were performed by using a ChemiDoc MP and the Image Lab software (Bio-Rad Laboratories, Hercules, California, CA, USA).