Comprehensive Western Blot Analysis

Western blot analysis was performed as previously described^{17 (link)}. Cells were lysed using radio-immunoprecipitation assay (RIPA) buffer (150 mM NaCl, 10 mM Tris, pH 7.2, 0.1% SDS, 1.0% Triton X-100, 1% sodium deoxycholate, and 5 mM EDTA). Samples were separated on 12% SDS polyacrylamide gels and transferred onto PROTRAN nitrocellulose membranes (Shleicher and Schuell Co.). After blocking with PBS containing 5% nonfat dry skim milk and 0.07% (vol/vol) Tween 20, the membranes were incubated with antibody specific for β-catenin (1:1000, sc-7963, Santa Cruz Biotechnology, Inc.), CXXC5 (1:500, Lab made), PPARγ (1:1000, ab19481, Abcam), or C/EBPα (1:1000, 2295, Cell Signaling Technology) at 4 °C overnight. Membranes were then incubated with horseradish peroxidase-conjugated anti-rabbit (1:1000, 170-6515, Bio-Rad) or anti-mouse (1:1000, 14790, Cell Signaling Technology) IgG secondary antibody. Protein bands were visualized with enhanced chemiluminescence (GE Healthcare) using a luminescent image analyzer, LAS-3000.