Western blot analysis

Cells in culture were lysed using Complete Lysis-M kit (Roche). The protein concentrations of the lysates were quantified using Coomassie Plus Protein Assay Reagent (Thermo Scientific). 10 ug of protein for each sample were loaded on to 8% SDS-PAGE gel. Separated proteins were transferred to a PVDF membrane (L-08008-001, Advansta) in wet transfer buffer. Membranes were blocked with 5% milk in TBST (0.5%) for 1 hour at room temperature before incubation at +4°C overnight with the following antibodies: HER2 (1:1000, ab8054, Abcam), total AKT (1:1000, sc8312, SantaCruz), phospho-AKT (Ser 473, 1:1000, sc-7985-R, SantaCruz), total ERK2 (1:1000, sc-154, SantaCruz), phosho-ERK 1/2 (Thr 202 / Tyr 204, 1:1000, sc-81492, SantaCruz) and beta-actin (1:1000, 634801, Biolegend) in 3% milk powder-TBST. After incubation with HRP-conjugated secondary antibodies, the protein bands were detected using WesternBright Sirius Kit (K-12043-D20, Advansta).