Western Blot Analysis of Key Signaling Proteins

Cell lysates were prepared using NP-40 lysis buffer as previously described [16 (link)]. The lysate was centrifuged at 14,000× g for 15 min at 4 °C. Bicinchoninic acid (BCA) assay was used to normalize protein concentrations across the samples, and the proteins were resolved on a 7.5–12% SDS-PAGE gels. Western blot analysis was carried out with primary antibodies specific to the proteins of interest as followed: β-catenin (sc-133240, Santa Cruz Biotech, Dallas, TX, USA), AMPKα (#2603, Cell signaling, Danvers, MA, USA), phospho-AMPKα (#2535, Cell signaling, Danvers, MA, USA), β-actin (ab8227, Abcam, Cambridge, MA, USA), AXIN1 (#06-1049, Sigma, St. Louis, MO, USA). Chemiluminescence detection of proteins was carried out and images were captured using an Odyssey-Fc Imaging System (LI-COR Biosciences, Inc. Lincoln, NE, USA).