Anti β catenin e 5

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).

Proteins extracted from fibroblast homogenates were separated by 10% SDS–polyacrylamide gel electrophoresis and then transferred to a 0.45 μm pure nitrocellulose membrane (Bio-Rad) by transfer buffer. The blots were blocked with milk at 5% for 1 h and incubated overnight at 4 °C with the monoclonal antibodies: anti-Human N-cadherin (1 mg/mL) (R&D system), anti-vimentin cloneV9 (1:250) (SIGMA), anti-a-actin (N-19) (1:200) (Santa Cruz), anti E-cadherin (G-10) (Santa Cruz) and anti-cytokeratin, pan (mixture) C2562 (SIGMA), anti-β-catenin (E-5) (Santa Cruz) and anti-MMP-9 (626–644) (Ab-3) (Calbiochem). Primary antibodies-bound membranes were incubated at 4 °C overnight with HRP-conjugated secondary antibody (1:4000) anti-mouse (SIGMA). Further details are reported in a previous study [19 (link)]. For detection, an ECL chemiluminescence system (Therma Scientific) was used.