Bca method

Tissues and cells were lysed firstly, and protein content was measured using BCA method (Nanjing Jiancheng, China). 15 μg protein samples were separated on 8% SDS-PAGE and transferred onto nitrocellulose membrane. After blocking with 5% fat free milk for 30 min, the membranes were cultivated with primary antibodies (1:1000) at 4° C overnight. After washing with PBS, the membranes were cultured with secondary antibody (1:1500). Then, immune-reactive proteins were measured. The protein bands were analyzed through ImageJ software.

The liver at −80°C was used. The cytokine TGF-β1 (with the dilution ratio of antibody concentration being 1 : 1000) in liver was detected, and β-actin was used as a normalize control for protein loading. Total protein was extracted from liver samples by homogenization with RIPA extraction buffer (Servicebio, Wuhan, China). The protein concentration of liver samples was measured using a BCA method (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). Protein samples were then subjected to 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis separation (SDS-PAGE) and transferred to nitrocellulose membranes. Membranes were blocked for 1 h using 5% bovine serum albumin (BSA)/phosphate buffered saline (PBS), after which they were probed overnight with appropriate primary antibodies at 4°C. Blots were then washed using PBS and probed using appropriate horseradish peroxidase-linked secondary antibodies. The membrane bands were displayed by chemiluminescence (micropores), and the strip intensity of each channel was quantified by ImageJ 6.0 software.

To lyse the LMHs (Beyotime, Shanghai, China), RIPA buffer was utilized, which consisted of a mixture of protease and phosphatase inhibitors. The extracted proteins were prepared in a standardized manner using the bicinchoninic acid (BCA) method (Jiancheng Bioengineering Institute, Nanjing, China). Equal amounts of total protein were combined with 5× DualColor Protein Loading Buffer (Fude Biological Technology Co., Ltd., Hangzhou, China) and heated at 99 °C for 5 min. Proteins were separated using 12% SDS-PAGE with 80 V for 20 min and 150 V for 40 min. Then, they were transferred to a nitrocellulose (NC) filter membrane in an ice–water mixture at 260 mA for 1 h. After 2 h of blocking in 5% skimmed milk at room temperature, it was incubated with primary antibodies (lddddd in Table 2) at 4 °C overnight. Following three washes with TBST, the secondary antibodies, HRP-conjugated goat anti-rabbit IgG (Abclonal, Wuhan, China), were incubated with the membrane at room temperature for 1.5 h. Imaging was achieved using the FDbio-Pico ECL kit (Fude Biological Technology Co., Ltd., Hangzhou, China). To standardize the expression of target proteins, β-actin served as the reference protein. Band intensities were quantified using ImageJ version 1.8.0 (Rasband, Bethesda, MD, USA).

The sample of kidney was homogenized and the supernatants were subjected to protein concentration determinations using bicinchoninic acid (BCA) method (Nan jing jian cheng bioengineering institute, Nanjing, China). Quantitative assessment of tumor necrosis factor (TNF-α), interleukin-6 (IL-6) and monocyte chemotactic protein-1 (MCP-1) protein levels were measured by commercial available ELISA kits (MultiSciences, Hangzhou, China). Results were expressed as pg/mg protein. All determinations were performed blindly with respect to treatment.