Tanon 5200 chemiluminescent imaging system

Radioimmunoprecipitation assay (RIPA, Thermo Fisher Scientific, Waltham, MA, USA) lysis buffer containing PMSF was employed to separate total protein on ice for 37°C. After centrifugation at 12,000 × g for 20 min at 4°C, the concentration of the protein was then quantified using BCA Protein Assay Reagent Kit (Beyotime, Shanghai, China). Equal amounts of protein for each sample were loaded and separated on a 10% sodium dodecyl sulphate-polyacrylamide gel (SDS-PAGE) before being transferred onto a polyvinylidene difluoride (PVDF, Roche Diagnostics) membrane. After blocking in 5% skim milk powder at room temperature for 1 h, the membrane was incubated by primary antibodies overnight at 4°C. The primary antibodies were RAB18 (1 : 1000, Santa Cruz Biotechnology, Santa Cruz, CA, USA), E-cadherin, N-cadherin, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Next, horseradish peroxidase- (HRP-) conjugated secondary antibody (1 : 5000) was conducted to incubate the membrane at room temperature for 1 h. The Enhanced Chemiluminescence (ECL) Kit (KeyGen Biotech, China) was applied to measure the bands and imaged on a Tanon-5200 Chemiluminescent Imaging System (Millipore, Bedford, MA, USA).

Total cellular protein was extracted from cell lines or tumor tissues with radio-immunoprecipitation assay lysis buffer (P0013C) purchased from Beyotime Biotechnology (Shanghai, China). Nuclear proteins were separated with Nuclear Plasma Separation Kit (P0028, Beyotime Biotechnology). The concentration of protein was quantified using a BCA Kit (P0012, Beyotime Biotechnology). The cell lysate was then mixed with 5× loading buffer (P0015, Beyotime Biotechnology) and separated using SDS-PAGE with gels ranging from 8% to 12%. After transferring the proteins onto poly(vinylidene fluoride) membranes (Millipore, Danvers, MA, United States) and blocking with 5% BSA at room temperature for 1 h, the membranes were incubated with primary antibodies overnight at 4°C. The membranes were then incubated with secondary antibodies for 1 h and visualized in Tanon 5200 Chemiluminescent Imaging System (Shanghai, China) using an ECL Detection Kit (Millipore). Relative protein expression was normalized with Lamin B1 for the detection of NF-κB p65 or β-actin for the detection of the whole protein lysate and analyzed with Image J 1.52a (NIH, Bethesda, MD, United States).