Primary antibodies against e cadherin

The fetal bovine serum (FBS) was purchased from Gibco (Grand Island, NY, United States). BCA protein assay kit (No. P0011) and One Step TUNEL Apoptosis Assay Kit (No. C1089) were purchased from Beyotime (Shanghai, China). Transwell assay inserts (No. 354483) were purchased from Corning (Corning New York, NY, United States). Enhanced Chemiluminescence (ECL) was purchased from Thermofisher (Waltham, Massachusetts, United States). 1% crystal violet solution (No. V5265) was purchased from Sigma (St. Louis, MO, United States). Anti-rabbit IgG, HRP-linked Antibody, primary antibodies against E-cadherin, Vimentin, p-AMPKα, p-p44/42 MAPK, p-MEK1/2, p-AKT, AKT, p-mTOR, mTOR, p-IκBα, IκBα, p-NF-κB p65, NF-κB p65 and GAPDH were purchased from Cell Signaling Technology (Danvers, MA, United States). Primary antibody against Wnt 3a was purchased from Abcam (Cambridge, United Kingdom). The catalogue numbers of antibodies were shown in Supplementary Table S1. FITC Annexin V Apoptosis Detection Kit (No. 556547) and DNA Reagent Kit (No. 340242) were purchased from BD Biosciences (San Diego, CA, United States). Cell Titer 96^® AQueous One Solution Cell Proliferation Assay (MTS, No. G3581) was purchased from Promega (Madison, WI, United States). The macroporous resin (No. M0032) was purchased from Solarbio Life Science (Beijing, China).

The procedures for western blotting analysis and protein visualization were executed as described previously [24 (link)]. Primary antibodies against DVL3 and β-catenin were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Primary antibodies against E-cadherin, ZO-1, N-cadherin, Vimentin, Snail, CD44, CD133, SOX2, c-Myc were gained from Cell Signaling Technology (Danvers, MA, USA). Anti-β-actin (Santa Cruz, CA, USA) was used as a loading control. The horseradish peroxidase (HRP)-conjugated secondary antibody was obtained from ZSGB-bio (Peking, China). The signals were checked by an enhanced chemiluminescence detection kit(Invitrogen, Carlsbad, CA, USA) and Bio-Rad Molecular Imager (Hercules, CA, USA). The relative expression of above proteins was normalized against β-actin using Image J analysis.

The MGC‐803 cells were seeded into 6‐well plates. Total protein was extracted from treated cells using IP lysis butter. Subsequently, the protein was subjected to SDS‐PAGE separation and electro‐transferation to PVDF membranes. Primary antibodies against E‐cadherin, N‐cadherin, vimentin, Snail, and GAPDH were purchased from Cell Signaling Technology. All the secondary antibodies were obtained from PerkinElmer, Inc.

After the separation by sodium dodecyl sulphate‐polyacrylamide gel (SDS‑PAGE), the cellular protein was transferred to polyvinylidene fluoride (PVDF) membranes (Invitrogen Life Technologies). Followed the blocking with 5% fat‑free milk, the membrane was incubated with primary antibodies against E‑cadherin, vimentin, Wnt5a, p‐β‐catenin, β‐catenin, Met, C‐Jun and GAPDH (all from Cell Signaling Technology, Inc) overnight at 4°C and then incubated with a secondary antibody for 1 hours. Enhanced chemiluminescence kit (Beyotime Institute of Biotechnology) was used to visualize the blots.