Anti e cadherin

Cells were homogenized and lysed using radioimmunoprecipitation assay lysis buffer (100 mM NaCl; 50 mM Tris-HCl, pH 7.5; 1% Triton X-100; 1 mM EDTA; 10 mM β-glycerophosphate; 2 mM sodium vanadate; and protease inhibitor). Protein concentrations were detected by using the Bio-Rad protein assay reagent (Bio-Rad Laboratories, Hercules, CA, USA) according to the manufacturer’s instructions. Forty micrograms of protein was separated by 10% SDS-PAGE and transferred onto a nitrocellulose membrane using a commercial semidry blotting apparatus (Bio-Rad, Richmond, CA, USA). The immunoblot was incubated for 1 h with blocking solution (5% skim milk) at room temperature. Next, the membranes were incubated overnight at 4°C with the following primary antibodies: anti-CUL4B (1:500 dilution), anti-E-cadherin (1:500 dilution), anti-N-cadherin (1:1,000 dilution), anti-vimentin (1:500 dilution), anti-β-catenin (1:1,000 dilution), anti-c-Myc (1:500 dilution), and anti-cyclin D1 (1:200 dilution) (all Santa Cruz Biotechnology, Santa Cruz, CA, USA). After washing with TBS-T, the membranes were incubated at room temperature for 1 h with a 1:2,000 dilution of horseradish peroxidase-conjugated immunoglobulin G (IgG) (Santa Cruz Biotechnology). Target protein was detected by an enhanced chemiluminescence substrate kit (Pierce Biotechnology, Rockford, IL, USA).