Bca protein concentration determination kit

Total protein extracts were obtained by lysing frozen hepatic tissue (100 mg) using the Total Protein Extraction Kit (Beyotime Biotech). Protein concentrations were quantified by the BCA Protein Concentration Determination Kit (Beyotime Biotech). Proteins (40 μg) were separated by 10% sodium dodecyl sulphate (SDS) polyacrylamide gel electrophoresis and transferred onto nitrocellulose membranes (PALL). The membranes were blocked with 5% nonfat dry milk in TBST for 1 h at room temperature and then incubated at 4°C overnight with the corresponding primary antibodies (SIRT1, 1 : 1000, CST; Ac-NF-κB-p65, 1 : 200, CST; GAPDH, 1 : 1000, Beyotime). Then, bound antibodies onto membranes were detected by using the secondary antibody (1 : 10000). Subsequently, enhanced ECL reagent was used to visualize the membranes. Membranes were exposed and the band intensities were analyzed by Gel-Pro Analyzer 4.0 (Media Cybernetics, Rockville, MD, USA). The results are expressed as the ratio of SIRT1 and Ac-NF-κB-p65 to GAPDH densitometry.

Recombinant rat PEDF (GenBank accession number: NM_177927): Cusabio Biotech Co, Ltd (Wuhan, Hubei, China); The bacterial lipopolysaccharide (LPS) (L4391, O111:B4) and MPO colorimetric activity assay kits: Sigma (St. Louis, MO, USA). The LDH cytotoxicity assay kit and BCA protein concentration determination kit: Beyotime (Shanghai, China); Tissue and cell total protein extraction kits: Sangon Biotech (Shanghai, China). ELISA kits to measure TNF-α, IL-1β, and IL-6: Shanghai Renjie Biotechnology Co., Ltd. (Shanghai, China) [15 (link)]. ELISA kits to measure PEDF: Shanghai Yan Qi Biological Technology Co Ltd. The TUNEL kit, annexin V‑FITC/PI apoptosis detection kit, and DAPI staining solution: KeyGEN Biotech (Nanjing, Jiangsu, China). Anti-rabbit cleaved-caspase-3 (Catalog No. #9661), anti-rabbit p38 MAPK (Catalog No. #8690), anti-rabbit phospho-p38 MAPK (Catalog No. #4511), anti-rabbit NF-κB p65 (Catalog No. #8242), and anti-rabbit phospho-NF-κB p65 (Catalog No. #3033) antibodies: Cell Signaling Technology (Danvers, MA, USA). Anti-rabbit NLRP3 (Catalog No. ab263899), anti-rabbit PPAR-γ (Catalog No. ab178860) antibodies and PPAR-γ inhibitor (GW9662, Catalog No. ab141125): Abcam (Cambridge, MA, USA) [16 (link)]. Anti-rabbit RIP3 (Catalog No. 17563–1-AP) and anti-mouse β-tubulin (Catalog No. 66240–1-Ig) antibodies: Proteintech (Wuhan, Hubei, China).

The determination of pyruvate dehydrogenase (PDH), alpha-ketoglutarate dehydrogenase (KGDH), succinate dehydrogenase (SDH), and malate dehydrogenase (MDH) activity was carried out as previously described (Cheng et al., 2017 (link)). The activity of phosphoenolpyruvate carboxykinase (PEPCK) enzymatic activity was performed through the PEPCK Assay Kit according to the manufacturer’s manual (Suzhou Comin Biotechnology, Suzhou, China). V. alginolyticus cultured in medium with OFX was collected and washed three times with sterile saline. The bacterial cells were suspended in × 1 PBS (pH 7.0) to OD600 = 1.0. Samples of 30 ml were collected by centrifugation at 8,000 rpm for 5 min. Pellets were resuspended in phosphate-buffered saline (PBS), and the cells were lysed by sonication for 10 min (200 W total power with 35% output, 2 s pulse, 3 s pause over ice). The solution was then centrifuged with 12,000 rpm at 4°C for 10 min to remove insoluble materials. The protein concentration of the supernatant was quantified by the BCA protein concentration determination kit (Beyotime, P0009). Then, 200 μg proteins were used for the determination of enzyme activity.

Protein from cells was extracted using the RIPA buffer (Beyotime Institute of Biotechnology, product code: P0013C), was quantitated using a BCA protein concentration determination kit (Beyotime Institute of Biotechnology, product code: P0012S), and 20 µg of protein was separated by electrophoresis. Proteins were transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, product code: IPVH00010), which were blocked with 5% skim milk and then incubated with the primary antibody for 2 h at 25 ℃, followed by incubation with an appropriate secondary antibody for 1 h at room temperature. The primary antibodies were Higd-1a antibody (21,749-1-AP, 1:1,000, Proteintech Group, Inc., Wuhan, China) and GAPDH antibody (10,494-1-AP, 1:20,000, Proteintech Group, Inc.). The secondary antibody was anti-rabbit IgG which is an HRP-linked antibody (7074, 1:2,000, Cell Signaling Technology, Boston, Massachusetts, USA). A typically enhanced chemiluminescent kit (Thermo Fisher Scientific, Inc.) and ImageJ v1.48 (National Institutes of Health) were used for visualizing the membranes and densitometry, respectively, as previously described [26 (link)].

Approximately 20 μM ML241 and RV were added to MA104 cells grown in a dense monolayer in sequence, with MOI = 0.1. After 20 h of incubation, the cell surface was gently washed twice with PBS, and a high-efficiency RIPA cell lysis buffer (R0010, Solarbio) was used to extract the total cell proteins. The bicinchoninic acid (BCA) protein concentration determination kit (P0012, Beyotime, Zhengzhou, China) was used to determine the protein concentration, and then Western blotting was performed.

Protein was extracted by using Protein Extraction Kit (Millipore, Bedford, MD, USA) according to manufacturer's protocol. Protein concentration was determined by using a BCA protein concentration determination Kit (Beyotime, Beijing, China). Samples were incubated for 10 min at 95°C in boiling buffer (50 mM Tris, pH 8.8, 1% sodium dodecyl sulfate (SDS); 5% 2-mercaptoethanol, 10% glycerol, and 0.002% bromophenol blue). Protein samples were run on12% SDS- polyacrylamide gel electrophoresis (PAGE) and then transferred to nitrocellulose membranes. Membranes were then blocked with 5% nonfat milk (w/v) and incubated with rabbit anti-human anti-BDNF (1:1,000) or anti-GAPDH (1:3,000); and a goat anti-rabbit secondary antibody. Bands were analyzed with software Image J. 1.42q (National Institutes of Health, United States). GAPDH was used to normalize BDNF protein levels.