Quantitative Western Blot Analysis in Cell Lines

Cells lysis and western blot were carried out as previously described [18 (link)]. Antibodies for: ILK, Akt, E-cadherin, Vimentin, GSK-3β, phospho-GSK-3β (Y216), β-catenin (all Transduction Laboratories BD), AR, Src, phospho-Src (Y416), phospho-ERK (T202/Y204), phospho-Akt (S473), phospho-GSK-3β (S9), D₁, D₃ and phospho-eIF4E (S209) (all Cell Signaling Technology Inc.), N-cadherin (R&D) and β-actin, ZEB1, (all Sigma) SNAIL (ABGENT), Calnexin, HSP-90 (all Calbiochem) were used to detect indicated proteins. The presence of the primary antibody was revealed with horseradish peroxidase-conjugated secondary antibodies diluted 1:2000 (Cell Signaling Technology Inc) and visualized with an enhanced chemiluminescence detection system (Bio-Rad) as previously described [19 (link)]. β-Actin served as a loading control. All immunoblots were stripped with stripping buffer containing 25 mM glycine–HCl, pH 2, 1% (wt/v) SDS for 30 min, and incubated in antibody against β-actin (dilution, 1:3000; Sigma-Aldrich), which served as a loading control. To obtain quantitative results, immunoblots were scanned using the public domain ImageJ software (National Institute of Health). Each data point was normalized against its corresponding actin data point.