Chemiscope 3400 mini imaging system

Total protein was extracted from HSC LX-2 cells, HSC-T6 cells or liver tissues with 1X SDS sample loading buffer (250 mM Tris HCL pH 6.8, 10% SDS, 30% glycerol, 5% β-mercaptoethanol and 0.02% bromophenol blue). Protein concentration was determined using a BCA kit (Thermo Fisher Scientific, Inc.). The lysates (25 µg/lane) were separated via SDS-PAGE on 6, 10 or 12% gels, and subsequently transferred to a nitrocellulose membrane (EMD Millipore). Following blocking with 5% milk at room temperature for 1 h, membranes were incubated with primary antibodies at 4°C overnight. Subsequently, the membranes were incubated with a HRP-conjugated goat anti-Rabbit IgG secondary antibody (1:10,000; cat. no. D110058; Sangon Biotech Co., Ltd.) for 1 h at room temperature. The bands were visualized using an enhanced chemiluminescence kit (Thermo Fisher Scientific, Inc.) with a ChemiScope 3400 mini imaging system (Clinx Science Instruments Co., Ltd.). Densitometry was performed for each group using ImageJ software (v1.50b; National Institutes of Health). The following primary antibodies were used: Anti-α-SMA (1:1,000; cat. no. 19245; Cell Signaling Technology, Inc.), anti-Col1α1 (1:1,000; cat. no. 72026; Cell Signaling Technology, Inc.) and anti-GAPDH (1:5,000; cat. no. 8884; Cell Signaling Technology, Inc.), which was used as the loading control.