E cadherin

Using a radio-immuno-precipitation assay buffer (RIPA, Sigma-Aldrich, St. Louis, MO, USA) containing protease and phosphatase inhibitors, total protein was isolated from the DMSO (control) and reversine treatment groups. The concentration of the protein was measured using a BCA assay. Western blotting was implemented in the light of standardized procedures using specific antibodies: PCNA (1:1000; Abcam, ab18197, Cambridge, Cambs, UK), Ki67 (1:1000, Proteintech, 27309-1-AP, Chicago, IL, USA), Bax (1:1000, Cell Signaling Technology, 2772 s, Danvers, MA, USA), Bcl-2 (1:1000, Cell Signaling Technology, 3498 s, Danvers, MA, USA), E-cadherin (1:1000, immunoway, YT1454, Plano, TX, USA), MEK1 (1:1000, Cell Signaling Technology, 12671 s, Danvers, MA, USA), p-MEK1 (1:1000, Cell Signaling Technology, 9127 s, Danvers, MA, USA), ERK1/2 (1:1000, Cell Signaling Technology, 4695 s, Danvers, MA, USA) and p-ERK1/2 (1:1000, Cell Signaling Technology, 4370 s, Danvers, MA, USA). The internal control was β-actin (1:3000, Proteintech, 20536-1-AP, Chicago, IL, USA). Quantity One (Bio-Rad, Hercules, CA, USA) was used to acquire and analyze the chemiluminescent signals. All samples were performed in triplicate.