Horseradish peroxidase conjugated anti rabbit or anti mouse igg

Total proteins from PDCs were isolated using RIPA buffer (Sigma-Aldrich, St. Louis, MO, USA) containing a protease inhibitor cocktail (Roche, Mannheim, Germany) and phosphatase inhibitor cocktail (Roche), and protein concentration was determined using a Quick Start Bradford Protein Assay (Bio-Rad, Hercules, CA, USA). Aliquots containing 30 μg of protein were subjected to 10% SDS-polyacrylamide gel electrophoresis and electrotransferred to nitrocellulose membranes. The membranes were blocked with 5% nonfat dry milk in Tris-buffered saline containing 0.1% v/v Tween 20 and probed overnight at 4°C with specific antibodies against the following proteins: p-EGFR, p-RAF, RAF, p-MEK, MEK, p-ERK, ERK, p-Rb1, Rb1, P-AKT, AKT (Cell Signaling Technology, Beverly, MA, USA), and beta actin (Sigma Aldrich). Horseradish peroxidase-conjugated anti-rabbit or anti-mouse IgG (Vector, Burlingame, CA, USA) was used as a secondary antibody, and signals were detected by chemiluminescence using ECL Western Blotting Substrate (Thermo Scientific, Rockford, IL, USA) and visualized using LAS-4000 (Fujifilm, Tokyo, Japan).