Lung samples and cell lysates were prepared in cold RIPA buffer containing a protease inhibitor cocktail (Sigma). Equal amounts of proteins (40 μg) were separated by SDS-PAGE and then transferred to PVDF membranes. The membranes were blocked with 5% nonfat milk and incubated with the following primary antibodies: anti-NLRP3 (1:1,000, Cell Signaling Technology, CST, Inc., United States), anti-caspase-1 (1:1,000, ABclonal), anti-GSDMD (1:500, Abcam, Cambridge, MA, United States), anti-SIRT1 (1:1,000, CST), anti-NF-κB p65 (1:1,000, CST), anti-phosphorylated (p)-NF-κB p65 (1:1,000, CST), anti-IκB-α (1:1,000, CST), anti-Acetyl-NF-κB p65 (Lys310) (1:500, CST), anti-PCNA (D3H8P) (1:500, CST), anti-ICAM-1 (1:1,000, Servicebio), and anti-VCAM-1 (1:1,000, Servicebio) overnight at 4°C. After that, the membranes were incubated with HRP-conjugated secondary antibody for 1–2 h. The blots were detected with an enhanced chemiluminescence (ECL) system (Thermo Fisher Scientific). The protein band intensity was normalized to β-actin (1:3,000, Sigma) and expressed as a ratio of the control.
Anti caspase 1
Manufactured by ABclonal
Sourced in United States, China
Anti-caspase-1 is a laboratory reagent used in biochemical research. It is a specific inhibitor of the caspase-1 enzyme, which plays a role in the inflammatory response. This product can be used to study the function and regulation of caspase-1 in various experimental systems.
Automatically generated - may contain errors
Lab products found in correlation
Anti nlrp3, by ABclonal (4 mentions)
Anti il 1β, by ABclonal (3 mentions)
Anti gsdmd, by Abcam (2 mentions)
Anti bcl 2, by ABclonal (2 mentions)
Anti asc, by ABclonal (2 mentions)
Anti β actin, by ABclonal (2 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Ecl system, by Thermo Fisher Scientific (1 mentions)
Anti nlrp3, by Cell Signaling Technology (1 mentions)
Anti icam 1, by Wuhan Servicebio Technology (1 mentions)
Protease inhibitor cocktail, by Merck Group (1 mentions)
β actin, by Merck Group (1 mentions)
Anti tlr4, by Bioss Antibodies (1 mentions)
Anti caspase 3, by Wuhan Servicebio Technology (1 mentions)
Anti myd88, by Bioss Antibodies (1 mentions)
Anti bax, by ABclonal (1 mentions)
Anti β actin, by Santa Cruz Biotechnology (1 mentions)
Anti hspa8, by ABclonal (1 mentions)
Anti il 18, by ABclonal (1 mentions)
Anti nf κb p65, by Affinity Biosciences (1 mentions)
10 protocols using anti caspase 1
1
Investigating NLRP3 Inflammasome Signaling
2
Immunohistochemical Analysis of Apoptosis Markers
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Tissues were embedded using paraffin and cut into 4-μm-thick sections (n = 3 animals per group, 6 sections per animal). Sections were deparaffinized and rehydrated, then placed in a box filled with citric acid antigenic repair buffer (pH 6.0) in a microwave oven for antigen retrieval. Later, they were incubated with 3% H2O2 for 10 min at room temperature. After blocking with 5% BSA for 30 min, the tissue sections were incubated with primary antibody anti-Caspase1 (ABclonal, Wuhan, China; Catalog: A0964, diluted at 1:200), anti-Caspase3 (Servicebio, Wuhan, China; Catalog: GB11009-1, diluted at 1:200), anti-TLR4 (Bioss, Beijing, China; Catalogue: bs-20594R, diluted at 1:200), anti-MYD88 (Bioss, Beijing, China; Catalogue: bs-1047R, diluted at 1:200), anti-BAX (ABclonal, Wuhan, China; Catalog: A0207, diluted at 1:500), and anti-BCL-2 (ABclonal, Wuhan, China; Catalog: A0208, diluted at 1:500). The secondary antibody was incubated at room temperature. After PBS washing, the sections were monitored and captured under a microscope.
3
Spinal Cord and Astrocyte Protein Extraction
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Lysis and protein extraction of spinal cord tissues or astrocytes was performed using the RIPA lysate buffer (P0013; Beyotime). The concentration of the extracted protein was determined by the BCA Protein Assay Kit (P0011; Beyotime). The protein (20–40 µg per lane) was separated on 8–15% SDS-PAGE gel and transferred to polyvinylidene fluoride membrane (IPVH00010; Millipore, Billerica, MA, USA). After blocking with 5% (m/v) skim milk for 1 h at room temperature, the membrane was incubated at 4 °C overnight with primary antibodies in skim milk at a dilution of 1:1000. These primary antibodies were as follows: anti-HSPA8 (A14001; Abclonal), anti-NLRP3 (A5652; Abclonal), anti-ASC (A1170; Abclonal), anti-caspase-1 (A0964; Abclonal), anti-NF-κB P65 (AF5006; Affinity, Cincinnati, OH, USA), anti-phosphorylated NF-κB P65 (p-NF-κB P65) (AF2006; Affinity), anti-IL-1β (A16288; Abclonal), anti-IL-18 (A16737; Abclonal), and anti-β-actin (sc-47778; Santa Cruz). After washing with Tris-buffered saline-Tween 20 (TBST) buffer, the membranes were incubated with horseradish peroxidase (HRP)-conjugated goat anti-rabbit (1:3000 dilution; A0208; Beyotime) or mouse (1:3000 dilution; A0216; Beyotime) secondary antibody at 37 °C for 40 min. The membranes were visualized using a chemiluminescence kit (Shanghai 7sea biotech Co. Ltd.) and analyzed using Gel-Pro-Analyzer 4.0 (Media Cybernetics, Inc.).
4
Molecular Mechanisms of Pentobarbital-Induced Necroptosis
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Pentobarbital sodium was safeguarded in Harbin Medical Pharmacological Laboratory. BSA and rhTrx‐1 were purchased from R&D System. Penicillin/streptomycin solution, Hoechst and 2% TTC dyeing solution were purchased from Solarbio. ROS and Necrostatin‐1 was purchased from Sigma. Annexin V‐PE/7AAD assay was purchased from BD Biosciences. All the ELISA kits were purchased from Cusabio. Dapi dyeing solution and JC‐1 kit were purchased from Beyotime. DMEM, foetal bovine serum (FBS) and MEM/EBSS were purchased from Hyclone. The primary antibodies used for immunofluorescence staining: Rabbit polyclonal anti‐Iba‐1 (Wako), Goat polyclonal anti‐Iba‐1 (Abcam), Mouse monoclonal anti‐RIPK1 (Santa cruz), Goat polyclonal anti‐CD206 (R&D System) and Rabbit polyclonal anti‐CD16 (Bioss). All the second antibodies used for immunofluorescence staining were purchased from Abcam company. The primary antibodies used for Western blot analysis: anti‐RIPK1, anti‐RIPK3, anti‐MLKL, anti‐pMLKL and anti‐CCL2 (Abcam); anti‐NLRP3, anti‐ASC, anti‐caspase‐1, anti‐caspase‐3 and anti‐β‐actin (ABclonal); anti‐MMP‐9 (R&D System); and anti‐CD16 (Bioss). The second antibodies used or Western blot analysis was Alexa Fluor 800‐conjugated Goat‐anti rabbit (LI‐COR).
5
Protein Extraction and Western Blot Analysis
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Total proteins were extracted from macrophages with RIPA lysis buffer (Solarbio, Beijing, China) containing 1% protease inhibitor cocktail (Meilunbio, Shanghai, China) to prevent protein degradation. After the protein concentration was measured by BCA kit (Meilunbio, Shanghai, China), 5× loading buffer was added and boiled for 10 min to fully denature the protein. Equal masses of proteins were separated by 12.5% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) gels and transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, MA, USA). Membranes were incubated with primary antibodies overnight at 4°C after blocking with 5% non-fat powdered milk at room temperature for 2 h. The next day, membranes were washed with Tris-buffered saline with Tween 20 (TBST) three times before incubation with horseradish peroxidase (HRP)-conjugated secondary antibodies. Then, the membrane bands were detected using enhanced chemiluminescence reagent (Epizyme, Shanghai, China) and analyzed by ImageJ software (version 8, National Institutes of Health). The primary antibodies were as follows: anti-LC3A/B (1:1,500, Cell Signaling Technology, USA), anti-caspase-1 (1:1,500, Abclonal, China), anti-GSDMD (1:1,500, Abcam, UK), anti-NLRP3 (1:1500, Abclonal, China), and anti-β-actin (1:200,000, Abclonal, China).
6
Investigating Inflammatory Signaling Pathways
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Santa Cruz Biotechnology (Dallas, Texas, USA) provided the anti‐TIMD4 (Cat# SC‐390805), anti‐ADAM17 (Cat# SC‐390859), and TGF‐β1 (Cat# sc‐130348) antibodies. Affinity Biosciences (Cincinnati, Ohio, USA) provided the anti‐p‐NF‐κB (Cat# AF2006), anti‐IL‐6 (Cat# DF6087), anti‐IL‐10 (Cat# DF6894), and anti‐IL‐18 (Cat# DF6252) antibodies. ABclonal (Boston, MA, USA) provided anti‐NF‐κB (Cat# A16271), anti‐phosphorylated p38 MAPK (p‐p38 MAPK, Cat# AP0526), anti‐p38 MAPK (Cat# A14401), anti‐caspase‐1 (Cat# A0964), and anti‐IL‐1β (Cat# A16288) antibodies. Proteintech (Chicago, IL, USA) provided the anti‐TLR‐4 (Cat# 66350‐1‐Ig), anti‐TGF‐β1 (Cat# 21898‐1‐AP) antibodies, HRP‐conjugated GAPDH monoclonal antibody (Cat# HRP‐60004), and fluorescein‐conjugated AffiniPure Goat Anti‐Rabbit IgG (H+L) (Cat# SA00003‐2). MedChemExpress (Monmouth Junction, NJ, USA) provided TAPI‐1 (Cat# HY‐16657) and SB203580 (Cat# HY‐10256). Yiyuan Biotechnology (Guangzhou, China) provided ox‐LDL (Cat# YB‐002). Sigma–Aldrich (St. Louis, MO, USA) provided LPS (Cat# L2630).
7
Immunofluorescence Analysis of Neuroinflammation Markers
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Spinal cord sections were dewaxed, conducted to antigen retrieval (Improved Citrate Antigen Retrieval Solution, P0083, Beyotime Biotechnology, Shanghai, CHN), treated with 3% hydrogen peroxide for 10 min, blocked with immunofluorescence blocking solution (P0102, Beyotime Biotechnology, Shanghai, CHN, room temperature) for 1 h, and then incubated with primary antibody overnight at 4°C, subsequently, incubated with fluorescent secondary antibody at room temperature for 1 h and observed under a fluorescence microscope (IX73, Olympus Corp, Tokyo, JPN). The fluorescence intensities were analyzed using ImageJ 1.51j8 (National Institutes of Health, US). The following primary antibodies were used: anti-IL-1β (1:100, A19635), anti-GFAP (1:100, A0237), anti-NLRP3 (1:100, A5652), anti-caspase-1 (1:100, A0964), anti-Bcl-2 (1:100, A0208) and anti-GSK-3β (1:100, A6164) were from Abclonal (Wuhan, CHN); anti-NF-κB (1:100, BF8005) and anti-DHODH (1:100, DF3991) were from Affinity (Jiangsu, CHN). The secondary antibodies used for immunofluorescence analysis were goat anti-mouse IgG H&L (1:100, FITC, ab6785) and goat anti-rabbit IgG H&L (1:100, FITC, ab6717) were purchased from Abcam (Cambridge, UK).
8
Western Blot Analysis of NLRP3 and Caspase-1
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Cell or tissue lysate was resuspended in 5× SDS loading buffer, subsequently incubated at 100°C for 5 min and centrifuged at 12,000 × g for 10 min. Protein concentrations were detected using a BCA Protein Assay Kit (Thermo Fisher Scientific). A total of 20 μg of protein from the tissue or cell lysate was separated by SDS-PAGE gel (Thermo Fisher Scientific) and then transferred onto polyvinylidene difluoride (PVDF) membranes (Millipore). The membrane was blocked using 5% nonfat milk for 2 h at room temperature and then incubated with appropriate primary antibodies: anti-NLRP3 (Abclonal, Wuhan, China) (1 : 1,000) and anti-Caspase-1 (Abclonal, Wuhan, China) (1 : 1,000) in blocking buffer overnight at 4°C. Anti-β-actin (HuaBio, Shanghai, China) (1 : 2,000) was used as a loading control. After washing three times with PBST, the membranes were incubated with HRP-conjugated secondary antibodies for 1.5 h at room temperature. The bands were detected using an ECL kit (MultiSciences, Hangzhou, Zhejiang, China).
9
Antibodies for Immunoblot Analysis
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The following antibodies were used for immunoblot analyses in this study: anti-R rabbit polyclonal antibody directed against the R peptide (peptide sequence EDPDEETSSQAVKALREMAD), anti-BZLF1 (Santa Cruz #sc-53904), anti-BMRF1 (Millipore #MAB8186), anti-IRF4 (Santa Cruz #sc-56713), anti-EBF1 (Biotechne #AF5165), anti-CD11C (Cell Signaling #45581), anti-caspase 1 (Abclonal #A0964), anti-ENPP2 (Proteintech #14243-1-AP), anti-Runx1 (Cell Signaling #4336S), anti-RUNX3 (Cell Signaling #13089), anti-NFATc1 (Santa Cruz #sc-7294), anti-NFATc2 (Cell Signaling #4389), anti-FYN (Santa Cruz #sc-434), anti-TNFRSF9 (Cell Signaling #34594), anti-CD9 (Cell Signaling #13174), anti-HLA DR/MHC class II (Santa Cruz #sc—53319), anti-AHR (Biotechne #AF6185), anti-CYP1B (Proteintech #18505-1-AP), anti-phospho-ERK (Cell Signaling #9101), anti-Tubulin (Sigma #T5168), anti-actin (Sigma #A5441), anti-V5 (Santa Cruz #sc—58052), and anti LMP2A (Santa Cruz #sc-101314). The secondary antibodies used were Horseradish peroxide (HRP)- labeled goat anti-mouse antibody (Fisher Scientific# 31431, 1:5000), HRP- labeled donkey anti-goat antibody (Fisher Scientific # A16005, 1:5000), HRP- labeled goat anti-rabbit antibody (Fisher Scientific# PI32460, 1:10000), and HRP-labeled goat anti-rat light-chain specific antibody (Millipore# AP202P).
10
Hippocampus Protein Expression Analysis
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Western blotting analysis was performed by proteins extracted from brain tissues (n = 3 rats per group). Briefly, the hippocampus tissues were crushed and frozen at −80°C in a mortar. Radioimmunoprecipitation assay, phenylmethylsulfonyl fluoride, and a small amount of liquid nitrogen were added and ground into a uniform liquid state. The sample was transferred to an Eppendorf tube, incubated on ice for 15 min, and centrifuged by a cold centrifuge. Then, the target protein was separated and stored in the refrigerator at −80°C.49 (link) According to the protein concentration result, 5 × loading buffer was added to the total protein sample, denatured at 95°C for 10 min and stored on ice. During electrophoresis, the protein sample was added to the well, the power was turned on, the voltage adjusted to 80v for approximately 25 min, then increased to 120 v. After gel electrophoresis, the protein bands separated on the gel were transferred to Polyvinylidene fluoride (PVDF) membrane, and poly cloned with anti-Caspase1 (ABclonal, Wuhan, China; Catalogue: A0964, diluted at 1:200). The PVDF membrane was incubated with a horseradish peroxidase-labeled secondary antibody. Finally, the bands were visualized by enhanced chemiluminescence. Image J software was used to analyze the intensity of the bands.
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