Protein Extraction and Immunoblotting

Parts of the tumor specimen were extracted in a homogenization buffer containing 1% NP-40, 0.5% sodium deoxycholic acid, and 0.1% SDS, supplemented with a protease inhibitor cocktail (Roche Diagnostics GmbH, Mannheim, Germany) [24 (link)]. The total protein concentrations of lysates were measured by Bio-Rad Protein Assay (Bio-Rad, Hercules, CA, USA) using a series of bovine serum albumin as standards. A total of 50 μg per sample was separated on a homemade 10 or 12% SDS-polyacrylamide gel electrophoresis and then transferred onto nitrocellulose membranes and incubated with different primary antibodies (Table S1) overnight at 4 °C, followed by incubation with horseradish peroxidase-conjugated rabbit (#7074, Cell signaling, Danvers, MA, USA), goat (sc-2354, Santa Cruz, Dallas, TX, USA), and anti-mouse secondary antibodies for one hour at room temperature. The blots were then visualized using an enhanced chemiluminescence detection kit (PerkinElmer Life Sciences Inc. Waltham, MA, USA). Anti-β-actin monoclonal was used as a loading control. Finally, signal intensities were measured using the Luminescent image system (FUJIFILM, LAS-4000, Tokyo, Japan) and Multi Gauge 3.0 software (Fuji, Japan). Densitometry analysis of each bot was normalized by β-actin (n = 5 mice were analyzed in each group).