Phospho-EGFR and Phospho-Erk1/2 Western Blotting

Differentiated cells were treated with air (control) or WCS from 2 cigarettes with or without Gefitinib (500 nM) in media. Twenty-four hours later, cells were solubilized using 1% SDS, 10 mM Tris pH 8.3, 0.1 mM EDTA supplemented with cOmplete protease inhibitor cocktail and PhoSTOP phosphatase inhibitor cocktail (Roche, Indianapolis, IN). Following sonication to reduce viscosity and centrifugation (12,000 rpm, 15 min at 4°C), proteins were quantified by BCA assay (Thermo-Fisher, Waltham, MA). Equal amounts of protein (25 μg) from each sample was diluted in buffer (100 mM Tris, pH 6.8, 4% SDS, 20% glycerol, and 100 mM dithiothreitol), separated on a 4–15% Ready Gel Precast SDS–polyacrylamide gels (Bio-Rad, Hercules, CA), and transferred by electrophoresis to PVDF 0.45 μm membranes Amersham Hybond (GE Lifesciences, Marlborough, MA). Membranes were then blocked (5% BSA in 137 mM NaCl, 20 mM Tris pH 7.6, 0.1% Tween-20) rinsed, and incubated with anti-phosphoEGFR rabbit mAb or anti-phospho ERK1/2 rabbit mAb (Cell Signaling, Danvers, MA) and anti-β-Actin monoclonal antibody (clone AC-74, Sigma, St Louis, MO). The membranes were then incubated with HRP-coupled goat anti rabbit IgG (KPL, Gaithersburg, MD). HRP was detected using SuperSignal West Pico Chemiluminescent Substrate (Thermo-Fisher, Waltham, MA), images were acquired using Biorad ChemiDocXRS and intensity signals were quantified and analyzed with ImageLab software (Bio-Rad, Hercules, CA). Blots were stripped and probed with anti-β-Actin mouse monoclonal antibody clone AC-74 (Sigma, St Louis, MO) as a loading control.