Bca assay

Total protein was extracted from the renal cortex of rats and quantified with the BCA assay (MultiSciences Biotech Co., Ltd., Hangzhou, China). Then the protein was diluted with 5× loading buffer and denatured at 100°C for 5 minutes. Afterwards, the protein samples were electrophorised in SDS polyacrylamide gel and then transferred onto PVDF membranes (Millipore Corporation, Bedford, MA, USA). The nonspecific background bindings were blocked with 5% nonfat milk for an hour. Then the membranes were incubated in primary antibodies at 4°C overnight and secondary antibodies for 1 hour. Optical density of the bands was scanned by Odyssey infrared fluorescence imaging system (LI-COR, USA) and quantified using ImageJ (National Institutes of Health, USA). The primary antibodies used are as follows: Wnt4 (sc-376279, Santa Cruz Biotechnology, Inc., USA, 1: 500), TCF4 (sc-166699, Santa Cruz Biotechnology, Inc., USA, 1: 500), GSK3β (ab93926, Abcam, Inc., UK, 1: 1000), p-GSK3β (ab131097, Abcam, Inc., UK, 1: 1000), E-Cadherin (GTX100443, GeneTex Inc., CA, USA, 1: 1000), ɑ-SMA (ET1607-43, Hangzhou HuaAn Biotechnology Co., Ltd., Hangzhou, China, 1: 5000), and Vimentin (ab92547, Abcam, Inc., UK, 1: 5000).

Total protein was extracted from the lung tissue homogenate after RIPA lysis buffer addition (Multi Sciences, Hangzhou, China). Proteins were quantified using the BCA assay (Multi Sciences, Hangzhou, China) method. The proteins were denatured using a water bath at 100 °C for 5–10 min. The denatured proteins were isolated using 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto a polyvinylidene fluoride (PVDF) membrane (Millipore, Darmstadt, Germany). The membrane was blocked with 5% skim milk powder for 90 min and washed with Tris-Buffered Saline with Tween 20 (TBST) three times (10 min each time). Then, the membranes were incubated with primary rabbit anti-mouse monoclonal antibodies against RIG-I (CST, 4200S, 1:1000), MAVS (CST, 4983S, 1:1000), NF-κB p65 (CST, 8242S, 1:1000), and GADPH (CST, 2118S, 1:1000) at 4 °C. Then, the membranes were incubated with secondary antibody (horseradish peroxidase-conjugated goat anti-rabbit IgG; 1:5000; Multi Sciences, Hangzhou, China). Protein bands were detected using an electrochemiluminescence kit (Multi Sciences, Hangzhou, China), according to the manufacturer’s instructions and analyzed using ImageJ software.