Enhanced chemiluminescence detection reagent

Western blot was conducted as described previously.19 (link) Briefly, cells were lysed and quantified using Bicinchoninic Acid Protein Assay Kit (Beyotime Biotechnology, Shanghai, P.R. China). Protein lysates (15 µg) were separated by 10% SDS-PAGE and transferred to Millipore PVDF membranes. Blots were blocked with 5% non-fat dry milk in Tris-buffered saline buffer for 2 hours at room temperature and then incubated with diluted antibodies overnight at 4°C, followed by incubation with horseradish peroxidase-conjugated secondary antibody (Abcam, Cambridge, MA, USA) for 1 hour at room temperature. The blotting signal was visualized using an enhanced chemiluminescence detection reagent (Abcam). β-Actin served as a loading control.

Western blotting was conducted as described previously.16 (link) Briefly, cells were harvested and lysed in modified RIPA buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 mm EDTA, 1% Nonidet P-40, 0.25% sodium deoxycholate, and 1 mM sodium orthovanadate in the presence of protease inhibitors). Protein lysates (15 μg) were separated by 10% SDS-PAGE and transferred to Hybond-P PVDF membranes (Amersham Biosciences). Blots were blocked with 5% non-fat dry milk in Tris-buffered saline buffer for 2 h at room temperature and then incubated with diluted antibodies against various proteins for 2 h at room temperature, followed by incubation with horseradish peroxidase-conjugated goat-anti-mouse antibody (Abcam, Cambridge, MA) for 1 h at room temperature. The signal was visualized with an enhanced chemiluminescence detection reagent (Abcam, Cambridge, MA). The primary antibodies used were phosphoEGFR Y869 (6963; Cell Signaling), epidermal growth factor receptor (EGFR) (4267; Cell Signaling), phospho-EPHA2 Y772 (8244; Cell Signaling), EPHA2 (6997; Cell Signaling), E-Cadherin (3195, Cell Signaling), phosphoIGF1R Y1161 (ab39398; Abcam), (Insulin-like growth factor receptor 1) IGF1R (Ab39675; Abcam), and β-Actin (A5316, Sigma).