Anti e cadherin antibody

Cell lines were lysed in RIPA buffer. After boiling with sodium dodecyl sulfate loading buffer, equal amounts (10 µg) of the proteins were electrophoresed on 10% sodium dodecyl sulfate-polyacrylamide gels (SDS-PAGE) and transferred to Immobilon membranes (Millipore Inc., Bedford, MA, USA) by semidry blotting. Five percent milk powder dissolved in PBS buffer containing 0.1% Tween-20 was used to block non-specific binding. Then, using standard techniques, the membranes were probed with the following antibodies: anti-E-cadherin antibody (mouse monoclonal antibody to human E-cadherin; clone: HECD-1; dilution: 1:200; Takara, Tokyo, Japan), anti-ZEB1 antibody (rabbit monoclonal antibody to ZEB1; clone: D80D3; dilution: 1:100; Cell Signaling Technology, Danvers, MA, USA), and anti-β-actin antibody (mouse monoclonal antibody to β-actin; clone: C4; dilution: 1:1,000; Santa Cruz Biotechnology, Dallas, TX, USA). Antibody-antigen complexes were detected using Immobilon Western chemiluminescent horseradish peroxidase substrate (Millipore), and signals were recorded on a LAS-3000 mini system (Fujifilm, Tokyo, Japan).