Bca protein analysis kit

Mice lung tissue was homogenized with a mixture containing phenylmethylsulfonyl fluoride (PMSF), alkaline phosphatase inhibitors, and protease inhibitors (Solarbio, Beijing). Protein concentrations were determined with the BCA protein analysis kit (Solarbio, Beijing, China) according to its product instructions. Using a 10% (SDS-PAGE) gel, transfer the protein to a polyvinylidene fluoride (PVDF) membrane (MILLIBOLE, Billerica, MA). Block with skim milk powder for 2 hours at normal temperature, and incubated overnight at 4°C with primary antibodies including CYP2E1 (Affinity, Beijing, 1:1,000), Nrf2 (Cell Signaling Technology, Boston, 1:1,000), GAPDH (ABclonal, Wuhan 1:1000), CYP1A1 (ABclonal, Wuhan 1:1000), CYP2S1 (absin, Shanghai 1:1000), DNMT1 (ABclonal, Wuhan 1:1000), TET1 (ABclonal, Wuhan 1:1000), TET3 (ABclonal, Wuhan 1:1000) and then goat anti-rabbit IgG secondary antibody (Epizyme, Shanghai, China) was incubated at room temperature for 2 h. Detection was performed using the ECL system (Millipore, Billerica, MA, United States) and analysis was performed using ImageJ (NIH, United States) software.

After treatment for 24h, HUVEC were washed with ice-cold phosphate buffered saline (PBS). Briefly, total proteins of cultured cells were extracted with lysis buffer (RIPA, Millipore) with 1 mmol/L phenylmethylsulfonyl fluoride (PMSF), 1 mmol/L phosphatase inhibitor cocktail and 1 mmol/L protease inhibitor. The protein concentration was determined by a bicinchoninic acid (BCA) protein analysis kit (Solarbio, Beijing, China). The total lysate was separated using 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the separated proteins were transferred to polyvinylidene difluoride (PVDF) membranes. Membranes were blocked with QuickBlock™ Blocking Buffer for Western Blot (beyotime, Shanghai, China) for 20 min and then incubated with primary antibodies against the human phospholipase C Beta 1 Polyclonal antibody (PLC, 1:1000, Cat No. 26551-1-AP) and human ITPR1-specific Polyclonal antibody (IP₃R,1:1000, Cat No. 19962-1-AP) for 12h. GAPDH was used as an internal standard. Following incubation, PVDF membranes were washed with TBST three times for 10 min. Then PVDF membranes were incubated with peroxidase-conjugated specific secondary antibody for 1 h at room temperature. Bands were visualized by chemiluminescence, representative images were acquired, and Image J software (NIH Image, Bethesda, MD, USA) was used to quantify the density of each band.

The quantitative real-time PCR (qRT-PCR) was applied to detect the mRNA expression levels of SIRT1, NF-κBp65, IL-6, IL-10, and TNF-α in the lung tissues. QIAzol® Lysis Reagent (QIAGEN) was used to extract the total RNA. HiScript® II Q RT SuperMix was used to synthesize reverse transcription. ChamQ Universal SYBR and specific primers (Table 1) were used to perform the reactions (Table 2) with the application of Biosystems 7500 instrument. Both HiScript® II Q RT SuperMix and ChamQ Universal SYBR were purchased from Vazyme Biotech Co., Ltd. GenScript Biotech Co., Ltd. provided the primers.
Western blot (WB) was applied to detect the protein expression levels of SIRT1, acetylated NF-κBp65 (Ac-NF-κBp65), and NF-κBp65 in the lung tissues, crus lung tissue and adding RIPA lysis to extract the total protein. Before detecting protein expression, the protein concentration was detected first using the BCA protein analysis kit (Solarbio). Next, protein denaturation, electrophoresis, and transfer to polyvinylidene fluoride were performed. Blocking fluid was used to block cross-reacting antibodies, and then, the blotted membranes were incubated with primary antibody and stored at 4°C. The following day, the secondary antibodies were incubated. Finally, the protein bands were visualized using the Super ECL Plus reagent (Solarbio).

Cells or tissues were lysed by RIPA buffer added with 1 mM PMSF (Solarbio, China) and Protease Suppressor Cocktail (Thermo Fisher Scientific, United States) and cultivated on ices for 0.5 h. The protein concentrations were identified via the BCA protein analysis kit (Solarbio, China). The specimens were subjected to separation by 10% SDS-PAGE and moved to a PVDF film. Then, the treated films were subjected to 5% nonfat milk in TBST for 120 min under RT and immunoblotted with the specific primary antibody under 4°C nightlong. The films were subsequently cleaned with TBST and cultivated with alkaline phosphatase-conjugated second antibodies for 60 min under RT. Immunoblotting was observed via the Odyssey® Dlx Imaging System. ImageJ software was utilized to analyze the densitometric. The antibodies are presented in Supplementary Table S1.

Tissues were lysed with RIPA buffer supplemented with 1 mM PMSF (Solarbio, China) and placed on ice for 0.5 h. The protein concentrations were identified via a BCA protein analysis kit (Solarbio, China). The extracted proteins were separated by 10% SDS‒PAGE and transferred onto PVDF membranes (Millipore, Billerica, MA, USA). The following primary antibodies were used: RAB7A (Proteintech, Wuhan, China) and β-actin (Proteintech, Wuhan, China) antibodies. The following secondary antibodies were used: HRP-conjugated anti-rabbit IgG and anti-mouse IgG antibodies (Cell Signaling Technology, Danvers, MA, USA).

U251 was harvested after being treated with drugs. After centrifuged, total protein was extracted with RIPA buffer, and concentration was examined via BCA protein analysis kit (Solarbio, PC0020). Then the samples were separated by SDS-PAGE, and transferred onto polyvinylidene fluoride (PVDF) membranes. The membranes were incubated with primary antibodies overnight at 4°C, and incubated with appropriate peroxidase-conjugated secondary antibodies for 1.5 h at room temperature, and then visualized by enhanced chemiluminescence with imageQuant LAS 500 system.

Total proteins (30–50 µg) were isolated from renal tissue and HK-2 cells with RIPA buffer (Solarbio, Beijing, China) containing a protease-phosphatase inhibitor mixture. The proteins were collected by centrifugation at 12,000 r at 4 °C for 20 min, and the concentration was determined by a BCA protein analysis kit (Solarbio, Beijing, China). For immunoblotting, equal amounts of protein were resolved by SDS-PAGE, transferred onto polyvinylidene fluoride (PVDF) membranes (Burlington Millipore, MA, USA) and sealed with 5% skim milk at 37 °C for 1 h. Then, the blots were incubated at 4 °C overnight with primary antibodies against NQO1, Bax, Bcl-2, Nox1, Nox4, Cleaved Caspase-3, Sirt1 and β-actin. Incubation with secondary antibodies was performed at 37 °C for 1 h. After the blots were washed with TBS with Tween-20 (TBST), the bands were detected by an ECL reagent and scanned using a GE-Amersham Imager 600 (General Electric Company, USA). Band densitometry was assessed by National Institutes of Health (NIH) ImageJ 1.50 software.