2.6. Western Blotting Analysis of Proliferating Cell Nuclear Antigen (PCNA), Canonical NF-κB, and WNT Signaling Pathway

B16F10 and C26 cells and tumor tissues were lysed in tissue lysis buffer (T-PER) containing a protease and phosphatase inhibitor cocktail (Thermo Fisher Scientific) for 30 min on ice. Cell debris was removed by centrifugation at 15,294× g rcf (12,000 rpm) for 15 min at 4 °C. Protein concentration in the cell lysate was determined using a BCA protein assay kit (Pierce, Thermo Fisher Scientific). The proteins (10–20 µg) were separated by 10% SDS-PAGE and subsequently transferred onto a nitrocellulose membrane using the BioRad Turbo Trans system (Hercules, California, CA, USA). After blocking with 5% skimmed milk for 1 h in TTBS (20 mM Tris, 150 mM NaCl, and 0.05% Tween 20, pH 7.5), membranes were incubated overnight with 1:1000 diluted primary antibodies anti-PCNA (Abcam, Cambridge, MA, USA); anti-NF-κB and phospho NF-κB p65, anti-β-catenin, and anti-transcription factor T cell factor 1 (TCF1) (Cell Signaling, Danvers, MA, USA), and anti-Gapdh (Ambion, Thermo Fisher Scientific); as well as anti-mouse β-actin at 1:3000 dilution (Thermo Fisher Scientific). They were then incubated for 1 h with the respective secondary antibody (horseradish peroxidase-conjugated goat anti-mouse or goat anti-rabbit; Jackson Immuno Research, West Grove, PA, USA). Blots were developed using Super Signal West Pico (Thermo Fisher Scientific), and signals were detected with an Amersham Imager 600. Intensity of the bands was quantified by ImageJ densitometry, with β-actin as the loading control.