Bca protein quantity kit

Total protein was extracted from cells or tissues using RIPA cell lysis with protease inhibitor Cocktail and quantified by the BCA protein quantity kit (Beyotime, #P001). Mitochondrial protein extraction was performed using a mitochondrial isolation kit. The mitochondrial fraction was isolated using the Cell Mitochondrial Isolation Kit (Beyotime Biotechnology) according to the manufacturer’s instructions. The treated cells were first collected, 2.5 mL of mitochondrial isolation reagent was added, and then gently suspended for 15 min at 4 °C. The cell suspension was gently pulsed 10 times for 10 s each time with a homogenizer and continued to be centrifuged at 600 × g for 10 min at 4 °C, followed by 11,000 × g for 10 min at 4 °C. The residue was the mitochondrial fraction. Equivalent amounts of protein were separated by 7.5–12.5% SDS-PAGE and transferred to polyvinylidene difluoride membranes (0.45 μM PVDF, Millipore, USA). Then the membranes were blocked with skimmed milk for 60 min and incubated with the primary antibodies at 4 °C overnight. Next, the membranes were incubated with the corresponding HRP-conjugated secondary antibody for about 1–2 h at room temperature and the bands were visualized by ECL Western blotting substrate (Thermo Fisher Scientific, USA). The intensity of protein expression was measured using ImageJ software.

A YPR decoction consisting of seven herbs (prepared Rehmannia root 30 g, Eucommia ulmoides 15 g, Mulberry parasitism 30 g, Radix cyathulae 15 g, Apocynum 30 g, Puerarin 15 g, and Uncaria 15 g) was mixed in 300 mL of distilled water and extracted in a ceramic clay pot at 120°C. After boiling, it was extracted at 100°C for 30 min. Then 100 mL of the water extract was filtered through a sieve. Each of herbs was weighed according to the proportion and decocted until each milliliter contains 1.5 g drug. All herbs and decoctions were provided by the TCM pharmacy of the Putuo Hospital, Shanghai University of Traditional Chinese Medicine. Benazepril (Bena) was purchased from Novartis Pharmaceutical (Beijing, China).
Rats urine mAlb kits and urine α1-MG ELISA kits (XiTang Biotech Company Limited, Shanghai, China); rats TNF-α, IL-6, and IL-1 immunohistochemistry kits (Bohe Hengmai Biotech Company Limited, Shanghai, China); antibodies of NF-κB p65, IκBα, and GAPDH (Cell Signaling, Boston, USA); nuclear and cytoplasmic proteins extraction kits, BCA protein quantity kits, and RIPA lysate (Beyotime Institute of Biotechnology, Nanjing, China); rabbit anti-rat Histone H3.1 polyclonal antibodies (Signalway Antibody, Maryland, USA); and Luminescence agent (Millipore Corporation, Billerica, USA) were used.