Bca protein kit

Total protein of renal tissues and glomerular mesangial cells was extracted by RIPA lysis buffer supplemented with protease inhibitors (Solarbio, China). Protein concentrations were determined using the BCA Protein Kit (Solarbio, China). All samples were boiled at 95°C for 5 min. Equal amounts of proteins were separated in 8–12% SDS-PAGE gels and then transferred onto PVDF membranes (Millipore, United States). After blocked with 5% non-fat milk for 2 h, the membranes were incubated with various primary antibodies overnight at 4°C, namely rabbit anti-LC3B (1:1,500, GeneTex, United States), rabbit anti-P62 (1:1,000, Proteintech, China), rabbit anti-PINK1 (1:1,000, Abcam, United Kingdom), mouse anti-Parkin (1:200, Santa Cruz Biotechnology, United States), rabbit anti-Drp-1 (1:1,000, Cell Signaling Technology, United States), rabbit anti-Mfn-2 (1:1,000, Cell Signaling Technology, United States). And then the membranes were washed 3 times with Tris-buffered saline containing Tween-20 for around 15 min and subsequently incubated with corresponding secondary antibodies for 2 h. After washing 3 times with TBST for around 15 min, the bands were detected by using ECL reagents (Abbkine, United States) and their density was quantified by using ImageJ software.

Cells were lysed on ice for 20 min using RIPA extraction buffer (Beyotime, Shanghai, China) containing protease inhibitor. Proteins were collected and the concentrations were determined by BCA protein kit (Solarbio, Beijing, China). Then, protein lysates were separated by SDS-PAGE and transferred to PVDF membrane (Millipore, Darmstadt, Germany). The membranes were blocked with 5% defatted milk for 1 h and incubated with primary antibodies overnight at 4 °C. Subsequently, the membranes were incubated with HRP-conjugated secondary antibodies at room temperature for 2 h. The β-Actin expression was used as internal control for normalization. The brands were detected by enhanced ECL chemiluminescence reagent (Beyotime). Details of the antibodies used in experiments were listed in Additional file 1: Table S2.

The total protein in lung tissue was collected and total protein concentration was detected utilizing the BCA protein kit (pc0020; Solarbio). The PVDF membrane was blocked with 5% skimmed milk powder, and the primary antibodies ERK1/2 (4695T; CST, Danvers, MA, USA), p-ERK1/2 (4370T; CST), P65 (AF5006; Affinity), and p-P65 (AF2006; Affinity) were added and incubated overnight. The membrane was then rinsed and HRP incubated. The protein bands were detected by the ECL and protein gray value was calculated by Image J.

Through the use of RIPA lysis buffer (R0010, Solarbio, China), total protein was isolated from tissues and cells, followed by quantification using a BCA protein kit (PC0020, Solarbio, China). In the following step, protein lysates were separated on SDS-PAGE gel prior to being transferred into a polyvinylidene fluoride membrane. Subsequent to 2 hours of blocking, the membrane was added with primary antibodies (4°C, overnight) and secondary antibodies (ambient temperature, 1 hour) for incubation. On the iBright CL750 Imaging System (Thermo Fisher, USA), immunoblots were visualized using an ECL kit (PE0010, Solarbio, China). The antibody information is shown in Supplementary Table 4.

hUCMSCs cultured in 6-well plates were respectively pretreated with TMP (50, 100 μM) for 24 h following with or without NAC (1 μM) for 4 h. Then, cells were washed and exposed to H₂O₂ (500 μM) for 2 h. The protein was extracted by a protein extraction kit (Jiancheng Bioengineering, Nanjing, China) and quantified by a BCA protein kit (Solarbio Life Sciences, Beijing, China).
The concentration of MDA was determined using an MDA kit (Jiancheng Bioengineering). The quantified protein of cells together with mixtures provided in the MDA kit were kept in a boiling water bath for 1 h; then, the mixture was centrifuged and the supernatants were measured at 532 nm with a microplate reader (Molecular Devices, MA, USA).
The concentration of SOD was determined using an SOD kit (Jiancheng Bioengineering). The quantified protein of cells together with mixtures provided in the SOD kit were kept at 37 °C for 20 min; then, the total mixtures were measured at 450 nm with microplate reader (Molecular Devices).