Hrp conjugated anti rabbit immunoglobulin

Cells were lysed in RIPA buffer (Sigma-Aldrich) supplemented with inhibitors of proteinases and phosphatases (Complete Mini and PhosSTOP; Roche Diagnostics, Rotkreuz, Switzerland). The cell lysate with 20 μg of protein was electrophoresed in a 10–20% gradient polyacrylamide gel (DRC, Tokyo, Japan) and transferred to Clear Blot Membrane-p (ATTO, Tokyo, Japan). The blot was blocked using PBS with 0.1% Tween-20 containing 2% bovine serum albumin or 0.8% ECL Prime Blocking Reagent (GE Healthcare, Chicago, IL, USA). Primary antibodies employed were monoclonal anti-MAP kinase, activated (diphosphorylated ERK1 and 2) (clone MAPK-YT; 1:3000; Sigma-Aldrich), a monoclonal anti-p44/42 MAPK (Erk1/2) (clone 137F5; 1:1500; Cell Signaling Technology, Danvers, MA, USA), and monoclonal anti-beta actin (clone AC-15; 1:2000; Sigma-Aldrich). The secondary antibodies employed were a horseradish peroxidase (HRP)-conjugated anti-mouse immunoglobulin (1:30,000; GE Healthcare) or an HRP-conjugated anti-rabbit immunoglobulin (1:10,000; Cell Signaling Technology). These antibodies were diluted with Can Get Signal (TOYOBO, Osaka, Japan) according to the manufacturer’s instructions. Signals were visualized by the reaction with ECL Prime Detection Reagent (GE Healthcare) and digitally processed using an LAS 4000 mini-CCD camera system (Fuji Photo Film, Tokyo, Japan).

Cell lysates were harvested using cell lysis buffer, and an equal amount of each protein extract was resolved using 10% polyacrylamide gel and electro‐transferred onto 0.45‐μm hybridization nitrocellulose filter (HATF) membrane (Millipore, USA). Membranes were immunoblotted with goat polyclonal anti‐ACTIN antibody, rabbit monoclonal anti‐SNAIL antibody, rabbit monoclonal anti‐SOX2 antibody, rabbit polyclonal anti‐EGFR antibody, rabbit polyclonal anti‐pEGFR antibody, rabbit polyclonal anti‐AKT antibody, and rabbit polyclonal anti‐pAkt antibody (Cell Signaling, Danvers, Massachusetts, USA) overnight at 4°C. Membranes were immunobloted with either HRP‐conjugated anti‐rabbit immunoglobulin (Cell Signaling, Danvers, Massachusetts, USA) or HRP‐linked anti‐goat immunoglobulin (Santa Cruz Biotechnology, Dallas, Texas, USA) for 1 hour at room temperature. The protein signal was detected by enhanced chemiluminescence (Thermo, Waltham, Massachusetts, USA) using the Amersham Imager 600 (GE Healthcare Life Sciences, Chicago, Illinois, USA).