5.7. Western Blot Analysis

OSCC cells were lysed in RIPA buffer (pH 7.6, 50 mM Tris HCl, 300 mM NaCl, 0.5% Triton X-100, 2 μL/mL aprotinin) (Elpis-Biotech, Inc., Daejeon, Korea) mixed with 2 mM of PMSF and 2 μL/mL of leupeptin. The lysate was subjected to a Pierce BCA protein assay (ThermoFisher Scientific) for measuring the amount of protein, then separated in a SDS-PAGE gel and transferred onto nitrocellulose membranes (MilliporeSigma, Burlington, MA, USA), with the subsequent blocking step using 3% BSA. The primary antibodies used include: β-Actin, β-catenin, p-β-catenin, GSK3β, p-GSK3β (Santa Cruz Biotechnology, Dallas, TX, USA), Wntless (WLS) (Abcam, Cambridge, MA, USA) and Wnt3a (Cell Signaling Technology, Danvers, MA, USA). Chemiluminescence was detected using the WesternBright^TM ECL (Advansta Inc., San Jose, CA, USA) and WesternBright^TM Sirius kit (Advansta Inc.) together with the secondary antibody (Cell Signaling Technology). The signal intensity of each protein band was then quantified using a LAS 3000 (Fuji Photo Film Co., Ltd.).