Electrogenerated chemiluminescence

After 48-hour transfection, cell debris was removed by centrifugation, and total protein was extracted using RIPA cell lysis buffer. Protein concentration was determined by bicinchoninic acid (BCA) Protein Assay Kit (Pierce, Rockford, IL, USA) and proteins were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and blotted onto polyvinylidene difluoride membranes. The membranes were blocked with bovine serum albumin for 1 hour, and subsequently incubated with primary antibodies overnight at 4°C (Wnt1, 1:1,000; E-cadherin, 1:1,000; vimentin, 1:1,000; β-cadherin, 1:2,000; and N-cadherin, 1:400). After three washes with tris-buffered saline and Tween 20, the blots were incubated with secondary antibody conjugated to horseradish peroxidase (1:10,000) (Chemicon International, Inc., Billerica, MA, USA) at 37°C for 1 hour, and visualized by electrogenerated chemiluminescence (Amersham, Freiburg, Germany). The images were analyzed by Sigma Pro 5.0 software (SPSS Inc., Chicago, IL, USA). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal reference. Relative protein content = targeted protein gray value/GAPDH gray value. Samples in each group were analyzed three times.