Ab216330

Protein was extracted from cells by a protein extraction reagent (Roche, Switzerland), and measured by a BCA protein kit (Pierce Biotechnology, USA). 40-μg protein for each sample was electrophoresed in 10-15% SDS gel, then transferred to nitrocellulose membranes. After blocked with 5% BSA solution at room temperature for 1 h, membranes were incubated with primary antibodies at a dilution of 1/1000 at 4 °C overnight, including E-cadherin (#3195, cell signal technology, USA), Vimentin (#5741, cell signal technology, USA), Snail (#3879, cell signal technology, USA), c-MET (ab216330, Abcam, USA), total AKT (ab38449, Abcam, USA), p-AKT (ab8805, Abcam, USA), GSK3β (ab227208, Abcam, USA), p-GSK3β (#9322, cell signal technology, USA), or GAPDH (ab181602, Abcam, USA). Then, followed with secondary antibody (1:5000) at 37°C for 1h. The signals were detected by ECL Kit (Pierce Biotechnology, USA), and were analyzed by Image pro plus software.

After treatment with various compounds (0.2% DMSO served as a control), the cells were lysed with cell lysis buffer. The antibodies used were antibodies against Akt (9272S; CST, Danvers, USA), pAktSer473 (4060S; CST), Met (ab216330; Abcam, Cambridge, UK), β-catenin (8480S; CST), non-phospho (active) β-catenin (Ser45) (19807S; CST), non-phospho (active) β-catenin (Ser33/37/Thr41) (8814S; CST), MiTF (ab140606; Abcam), TRP2/DCT (ab221144; Abcam), and β-actin (9272S; CST), and the target bands were detected with a chemiluminescence system (Clinx Science Instruments, Shanghai, China).