Protein Expression Analysis in Breast Cell Lines

Analysis of the protein expression was performed as described previously [55 (link)]. Briefly, total cell lysates from breast cell lines were prepared in lysis buffer (20% SDS, glycerol, 1 M Tris (pH 6.8)) containing protease inhibitor cocktail (Sigma-Aldrich, USA). An equal amount of proteins (10 µg) were loaded and separated in 8% SDS-PAGE or 3–8% Tris–acetate gel (for CBP only) and then transferred into nitrocellulose membrane (Sigma-Aldrich, USA). Followed by immunoblotting with rabbit monoclonal primary antibodies against CBP (#7389), GCN5 (#3305), ERα (#13258) and HER2 (#4290) (Cell Signaling Technology, USA) at dilution 1:1000 and mouse monoclonal antibody against β-actin (#A5441) at dilution 1:2000 (Sigma-Aldrich, USA), then with secondary anti-rabbit IgG, HRP-linked antibody and anti-mouse IgG, HRP-linked antibody (Cell Signaling Technology, USA). The detection of membrane was carried out by the ECL method (Biorad, USA) and developed using ChemiDoc™ imaging system (Biorad, USA). The band quantification was carried out using Image Lab™ software (Biorad, California, USA) with β-actin as loading control.