Immunoblotting analysis of transduced cell lines

Transduced SKBR-3 and BT474 cells were cultured for 48 h and lysed using lysis buffer (Hepes pH 7.4 20 mM, NaCl 150 mM, 10% Glycerol, 1% Triton X-100) with protease inhibitors cocktail Complete (Roche Applied Science, Penzberg, Germany) and sodium orthovanadate or Phostop (Roche) used both as phosphatase inhibitors. Protein quantification was performed using Bradford protein assay (BioRad) and protein extracts were resolved on SDS-polyacrylamide gel electrophoresis (Invitrogen). Gels were then blotted onto nitrocellulose (GE Healthcare, Little Chalfont, UK) membranes and probed with appropriate primary antibodies (Table S3). Secondary antibodies were horseradish peroxidase-conjugated: anti-mouse or rabbit (Thermo Fisher Scientific) and anti-goat (Santa Cruz Biotechnology Inc.) and proteins detection was performed with ECL Detection Reagent (GE Healthcare) according to manufacturer's protocol. ECL signals were detected and measured by the Uvitec Chemiluminescence Imaging System and NineAlliance software (Uvitec Ltd., Cambridge, UK). ECL signals were detected and measured by the Uvitec Chemiluminescence Imaging System and ImageJ software (Bandyopadhyay et al., 2014 (link)).