Ecl western blotting analysis system procedure

Proteins were extracted from frozen tumors using RIPA buffer (50 mM Tris–HCl (pH 8), 150 mM NaCl, 0.5% deoxycholic acid, 0.5% triton) supplemented with protease and phosphatase inhibitors. Proteins were separated by SDS-PAGE and then electrophoretically transferred into nitrocellulose membrane and probed using the following primary antibodies: anti-GAPDH (V18 clone, 1/20000) purchased from Santa Cruz Biotechnology, anti-COX-2 (12282, 1/1000), anti-phospho Serin 473-AKT (4060, 1/2000), anti-PTEN (9552, 1/2000), anti-INPP4B (14543, 1/2000) and anti-phospho-S6 ribosomal protein (2211, 1/8000) purchased from Cell Signaling Technology (Ozyme). Proteins were detected according to the ECL Western Blotting Analysis System procedure (GE Healthcare, Buckinghamshire, UK). The intensity of the protein bands was quantified using ImageJ software.

Samples analysed in this study come from breast cell lines cultured under the conditions recommended by the suppliers, and authenticated in our laboratory by using the GenePrint 10 System kit (Promega, Madison, WI, USA). We perform authentication of our cell lines each 20 passages.
Methods are described in detail elsewhere [29 (link)]. Briefly, proteins were extracted from cell culture using TNMG buffer (20 mM Tris-HCl (pH 8), 150 mM NaCl, 5 mM MgCl₂, 10% glycerol, 0.5% NP-40, pH 8) supplemented with protease inhibitors. For the siRNA experiments, cells were seeded in p60 plates (500,000 cells per well), transfected with control siRNA or FOXO6 siRNA and the cellular extracts were prepared six days after transfection. The following antibodies were used in this study: anti-GAPDH (sc-20357, Santa Cruz Biotechnology, Santa Cruz, CA), used as internal control, anti-FOXO6 (19122-1-AP, Proteintech, Chicago, USA), and anti-cleaved PARP (9541, Cell Signaling, Beverly, MA, USA). Proteins were detected by the ECL Western Blotting Analysis System procedure (GE Healthcare, Buckinghamshire, UK).