Comprehensive Western Blot Analysis of Cellular Markers

Western blot was carried out as previously described [26 (link)]. Total proteins were extracted with lysis buffer (Beyotime Institute of Biotechnology, Shanghai, China) premixed with phenylmethanesulfonyl fluoride (PMSF) and phosphatase inhibitor (Roche). After samples were loaded into gels, electrophoresis, transferring and immunostaining were conducted. The primary antibodies used were: PCNA (1:2000), vimentin (1:1000), E-cadherin (1:1000), N-cadherin (1:1000), Nanog (1:1000), ERK1/2 (1:1000), phosopho-ERK1/2 (Thr202/Tyr204) (1:2000) (Cell Signaling Technology, USA), Tubulin (1:1000) and GAPDH (1:1000) (Beyotime, China). The immunoreactive bands were detected by enhanced chemiluminescence (New cell & Molecular Biotech, China).