Enhanced chemiluminescence detection kit

Total protein was extracted using RIPA buffer (Fdbio science, Hangzhou, China) containing 1% protease and phosphatase inhibitors (Bimake, USA). Protein quantification was performed using BCA Protein Assay Kit (Fdbio science, Hangzhou, China). Protein samples of 40ug from each group were separated by 4-20% SDS-PAGE (GenScript, Nanjing, China) and then transferred to 0.22 μm PVDF membranes (Millipore, USA). Following a 2-hour blocking step in 3% Bovine Serum Albumin (BSA) at room temperature, the membranes were incubated overnight at 4℃ with primary antibodies including anti‐β-actin (1:5000; YM3028, Immunoway), anti-MMP3 (1:1000, ab52915, Abcam), anti‐E‐cadherin (1:500; YT1454, Immunoway), anti‐ZO-1 (1:500; YN1410, Immunoway), anti-N-cadherin (1:500, YT2988, Immunoway), anti-β-Catenin (1:1000, YM3403, Immunoway), anti-Snail (1:500, YT4351, Immunoway) and anti-Vimentin (1:500, YT4880, Immunoway). The next day, the membranes was washed 3 times with TBST and incubated with HRP-secondary antibody for 2 hours at room temperature. Immunoblots were detected with an imaging system (Bio-Rad, USA) and an enhanced chemiluminescence detection kit (Fdbio science, Hangzhou, China). GAPDH was selected as a loading control.

Western blotting was performed as described previously [47 (link)]. Briefly, all tissue samples and HCC cells were lysed with RIPA buffer (Pioneer, Xi’an, China) containing protease inhibitors (Roche, Indianapolis, IN, USA). Protein concentration was quantified by a BCA Protein Assay Kit (Thermo Fisher Scientific, USA). Equal amounts of protein lysates were separated on 10–12% sodium dodecyl sulphate–polyacrylamide gels, Then, the proteins were transferred onto polyvinylidene difluoride membranes (Merck Millipore, Germany). After blocked in 5% skim milk 1 h at room temperature, the membranes were incubated with specific primary antibodies at 4 °C overnight. The following day, the membranes were washed thrice with TBST and incubated with the appropriate secondary antibody for 1 h. Protein bands were visualised using an enhanced chemiluminescence detection kit (Fdbio, Hangzhou, China). Imaging signals were acquired and analysed on ChemiDoc™ Touch (Bio-Rad, United States). The antibodies are listed in Table S2.

Human GC cells were lysed with precooled RIPA buffer (Pioneer, Xi’an, China) with protease inhibitor (Pioneer, Xi’an, China) for 30 min on ice. The samples were then centrifuged (14,000 rpm for 20 min at 4°C), supernatants were collected. The protein concentrations were determined by the BCA quantification kit (Fdbio, Hangzhou, China). Protein samples (20 μg) were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes (Merck Millipore, Germany). The membranes were blocked with 5% skim milk for 1 h at room temperature and incubated with specific primary antibodies at 4°C overnight. Before and after incubation with the secondary antibody for 1 h at room temperature, the membranes were thoroughly washed with TBST buffer containing Tween-20 (Fdbio, Hangzhou, China) for 10 min, a total of three times. Finally, the membranes were exposured with an enhanced chemiluminescence detection kit (Fdbio, Hangzhou, China) to show protein bands. GAPDH was performed as an internal control. Imaging signals were acquired and analyzed by ChemiDoc™ Touch (Bio-Rad, United States). The primary and secondary antibodies used are listed in Supplementary Table S2.