Whole-Cell Lysate Preparation and Western Blot Analysis

The whole-cell lysates were prepared with lysis buffer containing 150 mM NaCl, 3 mM NaHCO₃, 0.1% of Triton X-100 and a mixture of protease inhibitors (Roche Diagnostics, CA) plus 2 mM Na₄VO₄ and 5 mM NaF. The whole-cell lysates were homogenized with a Kontes’ Pellet Pestle, frozen in −80 °C for minimum 25 minutes and then separated from insoluble cell materials by centrifugation at 16,000 g in a bench-top Eppendorf centrifuge at 4 °C. Nuclear fractions were obtained as we described29 . Protein concentration was determined with the BCA system (Pierce, Rockford, IL). The Western blotting was performed as we previously described29 44 (link)60 (link). The primary antibody anti-β-catenin (E5) and anti-β-actin were purchased from Santa Cruz Biotechnology (Dallas, TX). NF-κB signaling was analyzed with NF-κB p65 antibody sampler kit (Cell Signaling, Danvers, MA). The second antibody anti-mouse IgG-HRP was purchased from Santa Cruz Biotechnology. The antibody-associated protein bands were revealed with Western blotting luminol reagent (Santa Cruz Biotechnology, Dallas, TX).