Horseradish peroxidase chemiluminescent kit

The transfected cells were lysed in protein lysis buffer (Beyotime Institute of Biotechnology, Nantong, Jiangsu, China) on ice for 30 min. The supernatants were collected after centrifugation for 15 min at 14,000 r/min and the concentration of the protein was calculated by the BCA Protein Quantification Kit (Beyotime Institute of Biotechnology). Proteins were separated by 10% SDS-PAGE gel and transferred onto a polyvinylidene difluoride membrane (Millipore, Billerica, MA, USA) in transfer buffer at 300 mA for 2 h. Then the membranes were incubated with Tris-buffered saline (TBS) containing 5% non-fat milk powder for 2 h at 4°C. The membranes were washed with TBS containing 0.2% Tween- 20 (TBST) three times, a nd then incubated with primary antibodies at 4°C overnight. Later, the membranes were washed with TBST three times and the specimens were hatched with secondary antibody in a secondary antibody solution for 2 h at room temperature. The indicated proteins were detected using a horseradish peroxidase chemiluminescent kit (Thermo Fisher Scientific) using an optional CCD camera and image processing system (Bio-Rad, Hercules, CA, USA). β-actin was used as a loading control. Moreover, N-cadherin, Vimentin, MMP9, AKT, phospho-AKT, mTOR, phospho-mTOR were bought from Cell Signaling Technology, Danvers, MA, USA. The secondary antibodies were also from Cell Signaling Technology.

Cancer cells were dissociated in RIPA lysis buffer (P0013D) (Beyotime, Haimen, China) and PMSF (ST506) (Beyotime). Split products were centrifuged at 18 500 g, and the supernatants were collected. A Bradford Protein Assay Kit (P0006) (Beyotime) was used to analyze protein concentration, and the proteins were subject to 10% SDS/PAGE electrophoresis. After separation the proteins were transferred onto poly(vinylidene difluoride) membranes (Sigma‐Aldrich), and the membranes were blocked with 5% skim milk (Guangming, Shanghai, China) and incubated with primary antibody at 4 °C overnight. Then, specimens were hatched with secondary antibody conjugated with horseradish peroxidase (Cell Signaling Technology, Beverly, MA, USA). Signals were detected using a horseradish peroxidase chemiluminescent kit (Thermo Fisher Scientific) using an optional CCD camera and image processing system (Bio‐Rad, Hercules, CA, USA). GAPDH (G8140, US Biological, Swampscott, MA, USA) was used as a loading control. SMURF1, DAB2IP and p‐RSK1 (Ser380) were purchased from Abcam. p‐AKT (Ser473), AKT, p‐mTOR (Ser2448), mTOR, p‐ERK (Thr202/Tyr204), ERK and RSK1 were obtained from Cell Signaling Technology.