Bca protein determination kit

In brief, the weighed liver and kidney samples were immersed in a 1 mL precooled RIPA (Solarbio, Beijing, China) buffer containing benzenesulfonyl fluoride (PMSF) inhibitor (Beyotime, Shanghai, China) and homogenized at 4°C. The homogenate was centrifuged at a speed of 12,000 r/min for 10 minutes, then the supernatant was collected, and the protein concentration of the sample was detected by BCA protein determination kit (Beyotime, Shanghai, China). Mix the protein sample with the sample buffer in a ratio of 4 : 1 and boil for 10 minutes. The mixture was separated from 10% SDS-PAGE gel and transferred to PVDF membrane (Bio-Rad, USA) for 1 hour. The membrane was sealed in 5% skim milk powder dissolved in TBST for 2 hours [21 (link)]. Then, the membrane was incubated overnight with anti-HPRT1 (1 : 1,000), PDZK1 (1 : 1,000), and GAPDH (1 : 8,000) antibodies at 4°C. After washing three times in TBST, the membrane was incubated in a suitable second antibody conjugated with HRP (Beyotime, Shanghai, China) for 2 hours at room temperature and then washed three times in TBST again. Protein bands were displayed by ECL kit (Beyotime, Shanghai, China) and analyzed by Image J software.

RIPA buffer and supporting PMSF (Solarbio Life Science, Beijing, China) were used to extract total protein content from Hepa cells, C2C12 cells, and liver and muscle tissues. Protein concentration was measured using the BCA protein determination kit (Beyotime Biotechnology, Shanghai, China). An equal mass (60 μg) of each protein sample was loaded into each well of an 8% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel. Proteins were then transferred to a 0.45 μm PVDF membrane, and nonspecific binding to the membrane was blocked by incubation in 5% (w/v) skimmed milk powder in1X TBST for 1 h. Membranes were then incubated with the indicated primary antibodies at 4°C overnight with shaking: anti-insulin receptor β, anti-phospho-insulin receptor β, anti-Akt, anti-phospho-Akt, and anti-β-tubulin. Antibodies were recovered, and membranes were washed three times for 5 minutes each with Tris buffer containing Tween 20 (TBST). The washed immunoblots were incubated with horseradish peroxidase-conjugated secondary antibody diluted in 5% skimmed milk powder in 1X TBST at room temperature with shaking for 1 h. Immunoblots were then visualized using the UltraSignal Ultra-Sensitive ECL Chemiluminescence Substrate (4A Biotech, Beijing, China), and β-tubulin staining was used as the loading control.

Luteolin and DTT were obtained from Aladdin (China). 1,4-butanediol diacrylate (BDD) was purchased from TCI (Shanghai, China). Dextran sulfate sodium (DSS, 35000 ​Da) was purchased from MP Biomedical (USA). Mouse Th1/Th2/Th17 CBA Kit, Cytofix/CytopermSoln Kit, Leuko Act Cktl with GolgiPlug, Transcription Factor Buffer Set, Fixable Viability Stain 780, FITC-Anti-CD4 antibody, BV605-Anti-IL-17A antibody, APC-Anti–INF–γ antibody and PE-Anti-IL-4 antibody were purchased from BD Biosciences (San Diego, USA). PE-Anti-FOXP3 antibody was purchased from eBiosciences (San Diego, CA). Myeloperoxidase (MPO) kit, glutathione (GSH) kit and ROS kit were supplied by Nanjing Jiancheng Bioengineering Institute (Nanjing, China). The BCA protein determination kit and Reactive Oxygen Species Assay Kit were purchased from Beyotime Biotechnology (China). Mouse Beta Actin antibody, Mouse Occludin antibody and Mouse ZO-1 antibody for western blotting were purchased from Proteintech (China). SYBR Premix Ex Taq™, Trizol reagent and PrimeScript™ RT 131 Master Mix were obtained from TaKaRa Bio (China). All other reagents are commercially available and can be used directly. All the solvents used were of analytical grade and were procured from Sinopharm (China).

Total protein of myocardial tissue was extracted, and the protein content was quantified using the bicinchoninic acid (BCA) protein determination kit (Beyotime Institute of Biotechnology, Shanghai, China). Equal amounts of protein samples were subjected to 12% and 8% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Then, the gels were transferred onto polyvinylidene difluoride membranes. Next, the membranes were immersed with 5% nonfat milk powder in TBST (Tris-buffered saline with 1% Tween-20) for 2 h. Membranes were incubated overnight at 4°C with mouse anti-LC3-II (1 : 500), p-MLC (1 : 2000), rabbit anti-MLC (1 : 500), p-MYPT1 (1 : 2000), MYPT1 (1 : 500), p-AKT (1 : 8000), AKT (1 : 1000), Beclin 1 (1 : 2000), and GAPDH (1 : 5000). After washing three times with TBST, the membranes were incubated with the corresponding horseradish peroxidase- (HRP-) conjugated goat anti-rabbit secondary antibody (1 : 8000) and goat anti-mouse secondary antibody (1 : 4000) for 1 h at room temperature. After three washes with TBST, the membranes were exposed to Bio-Rad electrophoresis (Bio-Rad Laboratories, Hercules, CA, USA). All band intensities were analyzed by ImageJ software.

Cells were collected and washed with PBS once. Total proteins were extracted from cell samples using NP-40 lysis buffer containing 40μg/mL protease inhibitor and phosphatase inhibitor. The protein concentration was determined using the BCA protein determination kit (Beyotime Biotechnology Co., LTD. Shanghai, China) according to the manufacturer’s instructions. The same amount of 40 μg protein was electrophoretic in SDS-PAGE 10% gel. It was then transferred to the PVDF membrane (Millipore, USA). After being closed with 5% skim milk for 1h, they were incubated with primary antibody E-cadherin (Abcam, 1:1000) and N-cadherin (Abcam, 1:1000) at 4°C overnight. TBST film washing 3 times for 10min each time. The secondary antibody conjugated with horseradish peroxidase was incubated at room temperature for 1h. TBST washed the film 3 times, 10min each time. Protein bands were observed by ECL assay kit.

48 h after siRNA transfection, the cells were washed twice with PBS. RIPA Lysis Buffer (BL504A, Biosharp, China) supplemented with protease and phosphatase inhibitors was added to the cells and lysed on ice for 15 min. The liquid was collected and centrifuged. Protein concentration was determined using BCA protein determination kit (P0012, Beyotime, China). Total protein was transferred to PVDF membrane (Millipore, Billerica, MA) after electrophoresis in 10% or 7.5% SDS-PAGE. After blocking the nonspecific sites on the membrane with 5% sealant for 1 h at room temperature, the membrane was applied to CASP4 (1:1000, #4450, Cell Signaling Technology, USA), NLRP1(1:1000, ab36852, abcam, USA), Vinculin (1:100000, V9264-100UL, Sigma-Aldrich), GSDMD (1:2000, TA4012, Abmart China), p-ERK (1:2000, #4370, Cell Signaling Technology, USA) and incubated overnight at 4 °C. Then, the membrane was incubated with HRP-Conjugated Affinipure Goat Anti-Rabbit IgG(H+L) (SA00001-2, Proteintech) at room temperature for 1 h. The blots were finally visualized with the ECL Chemiluminescence substrate (hypersensitive) (BL523B, Biosharp, China). The film was exposed and developed by X-ray in a darkroom. The film was scanned and the strips were and quantified by Image J (1.46R). Three repeated experiments were set up in each group.