Goat anti rabbit secondary antibody

After cell lysis, total protein was collected and run on 12% Bis-Tris gradient gels (Thermo Fisher Scientific) and electropho retically transferred onto polyacrylamide membranes (Thermo Fisher Scientific). Nonspecific binding sites were blocked by incubating the membranes for 1 hour at 37°C with 5% nonfat dried milk in Tris-buffered saline containing 0.05% Tween-20 (TBST). Membranes were incubated overnight at 4°C with the following primary antibodies: anti-S100A4 (1:1,000; Santa Cruz Biotechnology Inc., CA, USA), anti-p53 (1:1,000; Santa Cruz Biotechnology Inc.), anti-E-cadherin (1:400; Santa Cruz Biotechnology Inc.), and anti-β-actin (1:1,000; Abcam, Cambridge, UK). Then, membranes were additionally washed and incubated with a goat anti-rabbit secondary antibody (1:20,000; Abcam). Bands were visualized using an enhanced chemiluminescence system (ECL, Pierce, Rockford, IL, USA).

HepG2 cells in each group were lysed in RIPA buffer. The lysates were centrifuged at 12,000 × g for 30 min at 4°C. The protein concentration was then determined using the BCA Protein Assay Kit (Beyotime Biotechnology, Shanghai, P.R. China) according to the manufacturer’s instructions. The protein (50 μg) was separated in 12% SDS-PAGE gel and then transferred onto a polyvinylidene fluoride (PVDF) membrane (Thermo Fisher). The PVDF membrane was blocked in 5% nonfat milk (Yili, Beijing, P.R. China) at room temperature for 3 h, and then incubated with rabbit anti-human phospho (p)-Smad2, total (t)-Smad2, p-ERK1/2, t-ERK1/2, E-cadherin, N-cadherin, vimentin, Slug, MMP9, and GAPDH, followed by goat anti-rabbit secondary antibody (all from Abcam, Cambridge, MA, USA). The immunoblots on the membrane were visualized using Enhanced Chemiluminescence Kit (Thermo Fisher) and recorded by G:Box Chemi XL system (Syngene, Cambridge, UK).

Cells were lysed with ice-cold lysis buffer (Beyotime Institute of Biotechnology, Shanghai, China), and protein was separated with 12% sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) (Beyotime Institute of Biotechnology), which was then transferred onto polyvinylidene difluoride membrane (Thermo Fisher Scientific). The membrane was then incubated with PBS containing 5% non-fat milk (Yili, Beijing, China) overnight at 4°C. After washed with PBST for 3 times, the membrane was incubated with rabbit polyclonal anti-SIRT1 antibody (1:200; Abcam, Cambridge, MA, USA) or rabbit polyclonal anti-GAPDH antibody (1:200; Abcam) at room temperature for 3 h. After washed with PBST for 3 times, the membrane was incubated with goat anti-rabbit secondary antibody (1:10,000; Abcam) at room temperature for 1 h. The immunoreactive band was detected using the enhanced chemiluminescence system (Thermo Fisher Scientific), according to the manufacture's instruction. The protein expression was measured using Image Pro Plus software (Media Cybernetics, Rockville, MD, USA).

The cells were lysed using radioimmunoprecipitation assay lysis buffer (Beyotime Institute of Biotechnology). Protein concentration was quanitified using a bicinchonininc acid assay kit. Total protein (50 µg/lane) was separated by 10% SDS-PAGE, followed by transfer onto polyvinylidene difluoride membranes (Millipore, Billerica, MA, USA). The membrane was subsequently incubated with rabbit anti-human TNFAIP8 antibody (cat. no. ab64988; 1:100), rabbit anti-human MMP2 (cat. no. ab3715; 1:100), rabbit anti-human MMP9 antibody (cat. no. ab38898; 1:100), rabbit anti-human GAPDH antibody (cat. no ab9485; 1:100; all Abcam, Cambridge, UK) at room temperature for 3 h, and then with a goat anti-rabbit secondary antibody (cat. no. ab7090; 1:10,000; Abcam) at room temperature for 1 h, and then visualized using an enhanced chemilluminescence kit (Pierce; Thermo Fisher Scientific, Inc.).

Cultured cells of each group were harvested, and the total protein was extracted using the RIPA lysis method. The protein concentration was detected by the bicinchoninic acid (BCA) method, and adjusted to 4μg/μL. Then, the total protein was isolated through 12% SDS-PAGE, and transferred to a polyvinylidene fluoride (PVDF) membrane. The membrane was dyed with Ponceau’s stain liquid, washed with phosphate buffer saline with Tween (PBST) for 5 min, immersed in 5% skim milk for 2 h, and then added with YAP1, caspase 3, Bcl-2, Bax, and β-catenin (1:1000 each) primary antibody (Abcam, USA), and sealed at 4°C all night long. The membrane was cleaned to remove the primary antibody, and then it was added with goat anti-rabbit secondary antibody (1:5000, Abcam, USA), cultured at 37°C for 1 h, washed with PBS 3 times, 5 min/time, and developed in the dark. The liquid on the membrane was removed by a filter paper, and the membrane was made to be luminescent through electrochemiluminescence (ECL) for development. The protein band was scanned, and the gray value was evaluated through Quantity One software to calculate the protein expression. The relative expression of protein = the gray value of target protein band/the gray value of β-Actin protein band.