Western Blot Analysis of Tumor Proteins

Tumor tissues from the remaining 3-4 mice in each group were lysed using ice-cold buffer containing 1% Nonidet P-40, 0.1% SDS, and 0.5% sodium deoxycholic acid, supplemented with a protease inhibitor cocktail (Biokit Biotech, Inc., Miaoli, Taiwan). Nuclear extracts were prepared using a cell nuclear protein extraction kit (Biokit Biotech) as described previously 15 (link). Lysate protein concentrations were determined using the Bio-Rad Protein Assay (Bio-Rad), using a dilution series of bovine serum albumin as standards. Equal amounts of denatured proteins from every single animal were separated by SDS-PAGE, transferred to nitrocellulose membrane, incubated with different primary antibodies, then stored overnight at 4 °C. The membranes were washed with wash buffer, followed by incubation with horseradish peroxidase-conjugated goat anti-mouse IgG antibody at room temperature. Immuno-reactive bands were visualized using an enhanced chemiluminescence detection kit (PerkinElmer Life Sciences Inc., Waltham, MA, USA). Signal intensities were quantified using the Fujifilm LAS-4000 system and Multi Gauge 3.0 software (Fuji, Japan). Mouse primary antibodies used are described in Supplementary Table S2.