Tcf21

Western blotting was performed similar to methods previously described [18 (link)]. Briefly, RIPA lysis buffer (Beyotime Biotechnology, Shanghai, China) was used to extract total protein of cells, and the concentration was measured by BCA protein assay (Beyotime Biotechnology, Shanghai, China). After that, samples containing 25% loading buffer were boiled at 100°C for 10 min. Equal amount of protein from each sample was loaded on to the well and separated using SDS-PAGE, then transferred to a PVDF membrane. After blocking in 5% milk blocking buffer for 2 hours, membranes were incubated at 4°C overnight with various of primary antibodies then. The primary antibodies used were: TCF21 (1: 1000; Abcam), E-cadherin, N-cadherin, Snail, Vimentin, Twist, Kiss-1 (1: 300; all from Santa Cruz) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (1: 1000; Cell Signaling Technology). After washing in TBST, specific secondary antibodies were added for incubating for 2 hours at room temperature. Protein signals on the membrane were visualized with the use of enhanced chemiluminescence (ECL) reagent.