Horseradish peroxidise conjugated antibodies

The protein samples from the cells and liver tissues were extracted following standard protocols (Beyotime Biotechnology, Haimen, China), and the protein content was determined using a BCA Protein Assay Kit. Proteins were subjected to SDS-PAGE and then transferred to a nitrocellulose membrane. After blocking, the membranes were incubated for 1 h at room temperature or overnight at 4°C. The primary antibodies are listed in Supplementary Table 2. The blots were then incubated with horseradish peroxidise-conjugated antibodies for 2 h at room temperature at a 1: 2000 dilution (Beyotime Institute of Biotechnology, China). Protein expression was detected by the enhanced chemiluminescence (ECL) method and imaged with a Bio-Spectrum Gel Imaging System (UVP, USA). To eliminate variations due to protein quantity and quality, the data were adjusted to GAPDH expression (IOD of objective protein versus IOD of GAPDH protein).

Total cellular protein from the culture-activated HSC-T6 and LX2 cells and mouse livers were extracted following standard protocols (Beyotime Biotechnology, Haimen, China), and the protein content was determined using a BCA Protein Assay Kit from Beyotime Biotechnology. Proteins (50 μg/lane) were subjected to SDS- PAGE and then transferred to a nitrocellulose membrane. After blocking, the membranes were incubated for 1 h at room temperature or overnight at 4 °C. The primary antibodies are listed in Supplemental Table 2. The blots were then incubated with horseradish peroxidise-conjugated antibodies for 2 h at room temperature at a 1: 2000 dilution (Beyotime Institute of Biotechnology, China). Protein expression was detected by the enhanced chemiluminescence (ECL) method and imaged with a Bio-Spectrum Gel Imaging System (UVP, USA). To eliminate variations due to protein quantity and quality, the data were adjusted to GAPDH expression (IOD of objective protein versus IOD of GAPDH protein). However, the protein levels of p-Smad2/Smad2 were not adjusted to GADPH expression.