Western Blot Analysis of p53, Runx3, ABCG2 and Wnt Signaling–Pathway Proteins β-Catenin, Cyclin D1, and cMyc Protein-Expression Levels

Protein concentration was measured by with a BCA protein assay kit, Thermo Fisher Scientific). Samples (40 μg) from each group were separated by SDS-PAGE, blocked for 30 minutes with a TBST solution containing 5% defatted milk powder for 1 hour, and then incubated with the primary antibodies diluted by TBST solution containing 3% FBS. Primary antibodies were anti-p53 (1:1,000, ab131442; Abcam), anti-Runx3 (1:1,000, ab49117; Abcam), anti-ABCG2 (1:1,000, ab16051; Abcam), anti-β-catenin (1:1,000, ab226977; Abcam), anti-cyclin D1 (1:1,000, ab226977; Abcam), anti-cMyc (1:1,000, ab39688; Abcam), and β-actin (1:1,000, ab8227; Abcam). Next, samples were incubated with goat antirabbit IgG (1:2,000, ab6721; Abcam) for 1 hour. The signal on the membrane was detected by ECL, protein expression standardized by β-actin, and the gray scale scanned and quantified by ImageJ software.