Snai1

Total protein was extracted from the cells. Approximately 25 μg sample was processed by 12% polyacrylamide gel electrophoresis and then transferred to polyvinylidene fluoride membranes. The membranes were placed in a blocking solution and slowly shaken for 2 h at 37°C. After blocking, the membranes were mixed with primary antibodies to PUMA (ab33906, 1:1000, Abcam, Cambridge, UK), Bax (#33-6400, 1:500, Invitrogen Inc., Carlsbad, CA, USA), Bcl-2 (#MA5-11757, 1:1000, Invitrogen), E-cadherin (GTX100443, 1:500, GeneTex, Inc., Alton Pkwy Irvine, CA, USA), Vimentin (GTX100619, 1:1000, GeneTex), Snai1 (#14-9859-82, 1:500, Invitrogen), N-cadherin (ab70611, 1:2000, Abcam), CDC73 (#PA5-17159, 1:1000, Invitrogen) and GAPDH (ab8245, 1:2000, Abcam) overnight at 4°C. The membranes were then incubated with the secondary antibody to IgG labelled with horseradish peroxidase (ab6721, 1:1000, Abcam) for 2 h at 37°C. Protein signals were visualized using enhanced chemiluminescence. The protein levels were analyzed using ImageJ software. The relative expression of the target protein was obtained by calculating the ratio of optical density (OD) value of the target protein to that of an internal reference.