Western Blot Analysis of Breast Cancer Cell Signaling

As described (9 (link), 10 (link), 16 (link)), breast cancer cell lines were grown to log-phase, washed twice with ice-cold PBS, and lysed in SDS lysis buffer according to the manufacturer's protocol (Cell Signaling Technology, Danvers, MA). Protein concentration was quantified using the bicinchoninic acid reagent (Sigma). Cell lysates containing 25 μg of protein were separated by SDS-polyacrylamide gel electrophoresis (PAGE), and transferred to PVDF membranes (Millipore). Membranes were immunoblotted with antibodies against phospho-AKT (S473), total AKT, phospho-p70S6K (T389), total p70S6K, phospho-rpS6 (S235/ 236), total rpS6, phospho-ERK (T202/Y204), total ERK (all from Cell Signaling Technology), tubulin (Sigma), and actin (Millipore). Bound antibody was visualized using SuperSignal West Pico (Thermo Scientific, Waltham, MA) or ECL plus (GE Healthcare, Auckland, NZ) and the chemiluminescence detection system by Fujifilm Las-3000.