Western Blot Procedure for Protein Analysis

The Western blot procedure was performed as described in detail earlier [19 (link)]. Briefly, equal amounts of crude proteinaceous extracts from each cell line (three preparations harvested at different times during the continuous culture) were fractionated by SDS-PAGE on mini-PROTEAN TGX any-kD precast gels or TGX Stain-Free FastCast gels (BioRad, Munich, Germany) and blotted to PVDF membranes. The membranes were blocked with nonfat dry milk or bovine serum albumin and incubated with primary antibodies to E-cadherin (#610181, BD Transduction Laboratories, Heidelberg, Germany) or vimentin (clone V9, #V6630, Sigma-Aldrich, Steinheim, Germany), phospho-Smad2 (S465/S467) (#3101, Cell Signaling Technology, Frankfurt/Main, Germany), Smad2 (#1736-1, Epitomics, Burlingame, CA, USA), and GAPDH (Cell Signaling Technology) as loading controls.