G box chemi xx9 imaging system

Total proteins from transfected and untransfected HepG2 cells were extracted using RIPA buffer (Vazyme Biotech, China). BCA assay (Beyotime, China) was conducted to quantify the extracted total proteins. For isolation of the protein samples, 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was carried out followed by transfer to polyvinylidene fluoride membranes (0.45 μm) with 300 mA current for 90 min. After transfer, the membranes were blocked in 5% BSA at ambient temperature for 1 h and then incubated overnight using rabbit antibodies against GPC3 (Abcam, USA; diluted 1 : 1000) and a rabbit antibody against GAPDH (Sangon Biotech, China; diluted 1 : 5000) as an internal standard at 4°C to normalize the protein expressions. On the next day, the membranes were further incubated using goat anti-rabbit IgG-HRP (Sangon Biotech, China; diluted 1 : 5000) for 1 h and then subjected to protein visualization using enhanced chemiluminescence (Vazyme Biotech, China). The G:BOX Chemi XX9 imaging system (Syngene, UK) was used for protein visualization.

Liver, heart, brown adipose tissue, and white adipose tissue samples (20–50 mg) were homogenized in 200 μl of radioimmunoprecipitation assay buffer supplemented with 1 × protease inhibitor cocktail. Small intestine and colon samples were homogenized in 200 μl of radioimmunoprecipitation assay buffer supplemented with 5 mM EDTA, 1 mM PMSF, and 5 × protease inhibitor cocktail. Homogenates were centrifuged at 10,000g for 10 min at 4 °C. Proteins (50–100 μg) were fractionated on 4 to 12% bis–Tris polyacrylamide gels, transferred onto PVDF membranes, and visualized by Ponceau S staining. The GAPDH antibody was used at a 1:3000 dilution and incubated at 4 °C overnight. The catalase and NUDT7 antibodies were used at a 1:500 and 1:6000 dilution, respectively, incubating for 1 h at room temperature. Horseradish peroxidase–conjugated goat anti-rabbit IgG was used as the secondary antibody at a 1:22,500 dilution, incubating for 1 h at room temperature. Antibody signals were detected by chemiluminescence on a G:BOX Chemi XX9 imaging system (Syngene). For quantification, the antibody signal in each sample was normalized to the total protein loaded, as determined by the intensity of the Ponceau S stain, and expressed relative to male mice fed the CD. Densitometric analysis was conducted using ImageJ (https://imagej.nih.gov/ij/).

After transfection of miRNA-122 mimic and miRNA-122 inhibitor groups, the total protein of HUVECs cells was extracted by adding RIPA buffer (Vazyme Biotech, China). Protein samples were separated by SDS-PAGE and transferred to polyvinylidene fluoride membrane by polyacrylamide sulfate gel electrophoresis. After the transfer, the membrane was sealed with 5% Bowene Serum Albumin at room temperature for 1h and subsequently incubated overnight with the required primary antibody at 4°C. The next day, the membrane was incubated with Sangon Biotech (China, 1:500 dilution) for 1h, and the protein was observed by enhanced chemiluminescence (Vazyme Biotech, China). G:BOX Chemi XX9 imaging system (Syngene, UK) for protein visualization was used. Individual western blotting was performed with primary antibody, Bax, Bcl-2, Caspase-3 antibody (Abcam, USA, 1:1000 diluted), Hes1, Notch1, Vascular Endothelial Growth Factors [VEGF], CCNG1 and GAPDH antibody (Sangon Biotech, China, 1:500 diluted).