All western blotting procedures were performed as previously described (30 (link)) with the following primary antibodies: anti-PRX3, anti-NLRP3 (Abclonal Biotechnology, Wuhan, China), anti-gasdermin D (GSDMD), anti-caspase-1, anti-cleaved caspase-1, anti-IL-1β (all from Cell Signaling Technology, Boston, MA, USA), anti-IL-18, anti-PRX5 (Abcam, Cambridge, UK), PRX6 (Zen bio., Chengdu, China) and anti-β-actin (Bimake, Houston, TX, USA).
Anti nlrp3
Manufactured by ABclonal
Sourced in China, United States, United Kingdom
Anti-NLRP3 is a laboratory equipment product designed for the detection and analysis of NLRP3 (NLR Family Pyrin Domain Containing 3) protein. NLRP3 is a key component of the inflammasome, a multi-protein complex that plays a central role in the innate immune response. This product allows for the investigation of NLRP3 expression and function in various biological contexts.
Automatically generated - may contain errors
Lab products found in correlation
Anti il 1β, by ABclonal (6 mentions)
Anti caspase 1, by ABclonal (4 mentions)
Bca protein assay kit, by Beyotime (4 mentions)
Ripa lysis buffer, by Beyotime (4 mentions)
Anti il 18, by ABclonal (2 mentions)
Anti asc, by Santa Cruz Biotechnology (2 mentions)
Anti gapdh, by Proteintech (2 mentions)
Anti p62, by ABclonal (2 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Anti gasdermin d, by Cell Signaling Technology (1 mentions)
Anti caspase 1, by Cell Signaling Technology (1 mentions)
Anti cleaved caspase 1, by Cell Signaling Technology (1 mentions)
Anti il 18, by Abcam (1 mentions)
Anti il 1β, by Cell Signaling Technology (1 mentions)
Anti β actin, by Santa Cruz Biotechnology (1 mentions)
Anti asc, by ABclonal (1 mentions)
Anti hspa8, by ABclonal (1 mentions)
Anti nf κb p65, by Affinity Biosciences (1 mentions)
Ipvh00010, by Merck Group (1 mentions)
Gel pro analyzer 4, by Media Cybernetics (1 mentions)
14 protocols using anti nlrp3
1
Western Blotting for Inflammasome Proteins
2
Spinal Cord and Astrocyte Protein Extraction
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
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.).
3
Dose-Dependent Cytotoxicity of CdTe Quantum Dots
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
KUP5 cells (5 × 104) were cultured in a volume of 2 mL complete medium in the wells of a 6-well plate overnight and incubated with 0, 5, 50, and 500 nM CdTe QDs for 24 h. Next, the cells were washed twice with PBS and 50 μL of RIPA Lysis Buffer (Beyotime, Shanghai, China) and 0.5 μL PMSF (Beyotime, Shanghai, China) was added to each well. The protein lysate was incubated on ice for 30 min and then centrifuged for 10 min (12000 rpm, 4 °C). The protein supernatant was placed in a new centrifuge tube and stored on ice. The concentration of the protein samples was quantified using the BCA protein assay kit (EpiZyme, Shanghai, China) and aliquots containing equal amount of proteins were resolved by SDS-PAGE in sample loading buffer and transferred to 0.2 μm PVDF membranes. Next, the membranes were washed with TBST 3 times for 10 min each. Finally, the membranes were trimmed and incubated overnight at 4 °C with diluted primary antibodies. ECL luminous solution (Thermo Fisher Scientific, USA) was prepared as a 1:1 solution, and proteins were detected. The primary antibodies used in this study included anti–NF–κB, anti-NLRP3, anti-IκB, anti-pro-IL-1β+IL-1β, and anti-GAPDH (all from ABclonal Technology, Wuhan, China) and anti-pro caspase-1 + p10 + p12 (Abcam, UK).
4
Western Blot Analysis of INS-1 Cell Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total protein of INS-1 cells was extracted and lysed in RIPA lysis buffer (Beyotime, Shanghai, China) including the protease inhibitor (Roche, CA, USA). A BCA protein assay kit (Beyotime, Shanghai, China) was used to quantify proteins, and samples were mixed with 1 SDS sample buffer (50 mM Tris, pH 6.8, 2% SDS, 10% glycerol, 50 mM DTT, and 0.01% bromophenol blue). Proteins were separated in 12% SDS-PAGE, transferred onto the PVDF membrane, and immunoblotted with anti-GSDMD (1 : 1000, ABclonal, USA) and anti-NLRP3 (1 : 1000, ABclonal, USA) at 4°C overnight. Secondary antibodies (goat anti-rabbit IgG H&L (HRP), Abcam, USA) were applied for 2-3 h at room temperature, and membranes were developed via the BCA protein colorimetric assay kit (Beyotime, Shanghai, China). Developed protein bands were quantified by Tanon Gel Image System V2.0 program (Shanghai, China).
5
Immunofluorescence Staining of Lung and THP-1 Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Immunofluorescence staining was performed as previously described with minor modification [28 (link)] [PMID: 33,472,663]. The lung tissue or THP-1 cell sections were blocked with 1% bovine serum albumin (BSA) (Sangon Biotech, Shanghai, China) for 15 min and incubated with anti-CD68 (diluted 1:50; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), anti-CD86 (diluted 1:100; ABclonal Technology, Wuhan, China)), anti-iNOS (diluted 1:100; Affinity Biosciences, Changzhou, China), anti-NLRP3 (diluted 1:100; ABclonal Technology, Wuhan, China), anti-ASC (diluted 1:50; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) and anti-p65 (diluted 1:400; CST, Danvers, MA, USA) overnight. Subsequently, the slices were incubated with corresponding secondary antibodies (Cy3: goat-anti rabbit, diluted 1:200, Invitrogen, Carlsbad, CA, USA; FITC: goat-anti-mouse, diluted 1:200, Abcam, Cambridge, UK), followed by counterstaining with 4’, 6-diamidino-2-phenylindole (DAPI) (Aladdin, Shanghai, China). Fluorescence microscope (Olympus, Tokyo, Japan) was used to capture stained images.
6
Western Blot Analysis of Inflammatory Markers
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total protein was extracted from cell lysates. Western blot analysis was performed using standard methods [13 (link)]. Primary antibodies: anti-NLRP3 (A12694; ABclonal, China);anti-IL17A (A0688; ABclonal, China);anti-IL17-RA (A10052; ABclonal, China);anti-IL1β(A12688; ABclonal, China); anti-IL18(A1115; ABclonal, China); anti-oxidative phosphorylation (OxPhos) complexes (I-NDUFB8, A19732; II-SDHB, A1809; III-UQCRC2, A4366; IV-MTCO2, A11522 and ATP5J, A3751; ABclonal, China).
7
Protein Extraction and Western Blot Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
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).
8
Immunofluorescence Analysis of Kidney Tissue
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The kidney tissue sections (5 μm) were deparaffinized, rehydrated, and washed with PBS. Cells were fixed in 4% paraformaldehyde, incubated with 0.1% Triton X-100 (Beyotime), and washed with PBS. Tissue sections and cells were blocked in goat serum for 15 min. For TOLLIP-TLR2 and TOLLIP-TLR4 staining, tissue sections were subjected to incubation with anti-TOLLIP (ABclonal, China) and anti-TLR2 (NOVUS, USA)/anti-TLR4 (Santa Cruz, USA) antibodies (1 : 50 dilution) at 4°C overnight, followed by incubation with FITC-conjugated goat anti-rabbit IgG or Cy3-conjugated goat anti-mouse IgG (Beyotime; 1 : 200 dilution) at room temperature for 90 min. For cleaved caspase-3, NLRP3, and p65 staining, tissue sections or cells were subjected to incubation with anti-cleaved caspase-3 (Affinity, China), anti-NLRP3 (ABclonal), and anti-p65 (Affinity) antibodies (1 : 100 dilution) at 4°C overnight, followed by incubation with Cy3-conjugated goat anti-rabbit IgG (1 : 200 dilution) at room temperature for 60 min. Next, tissue sections or cells were subjected to dihydrochloride (DAPI) (Aladdin) nuclear staining. Images were captured using a fluorescence microscope (OLYMPUS).
9
Renal Fibrosis Protein Profiling
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The proteins were isolated from the frozen kidneys and the concentration was calculated by the Bradford method. Immunoblotting examination was performed as previously described [21 ]. In this study, the primary antibodies used were anti-Ecadherin (1:1000,Abcam,UK), anti-ColIII (11,000, Abcam, UK),anti-caspase1. (1:1000, Abcam,UK), anti-TGFβR1 (1:1000,Abcam,UK), anti-Sirt1 (11,000, Abcam, UK), anti-NLRP3 (1:1000, Abclonal, China), anti-IL-1β (11,000,Abclonal,China), anti-Acetylated-lysine (1:500,Santa Cruz,USA), anti-ASC (1:500, Santa Cruz, USA), anti-TGF-β1 (1500,Santa Cruz,USA), anti-Smad3 (1,1000,CST,USA), and anti-Gapdh (12,000, Proteintech, USA). Protein bands were visualized using the chemiluminescence system (Tanon) for the required time. Quantitative analysis was performed using ImageJ software.
10
Hepatic Protein Analysis by Western Blot
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Proteins of hepatic samples or cells were analyzed using standard western blotting techniques. The antibodies of anti-α-SMA (ab32575, Abcam), anti-TGF-β1 (SAB4502954, Sigma), anti-P65 (10,745–1-AP, Proteintech), anti-p-P65 (AF2006, Affinity), anti-iNOS (13120S, CST), anti-CD86 (19,589, CST), anti-CD163 (16,646–1-AP, Proteintech), anti-CD206 (24,595, CST), anti-TLR4 (19,811–1-AP, Proteintech), anti-NLRP3 (A5652, ABclonal), anti-AKT (10,176–2-AP, Proteintech), anti-p-AKT (4060S, CST), anti-HO-1 (43,966, CST), anti-SOD1 (10,269–1-AP, Proteintecch), anti-MDA (MDA11-S, ADI), or anti-CASP1 (24232S, CST) were used as primary antibodies. Anti-GAPDH (10,494–1-AP, Proteintech) or anti-TUBULIN (#2144, CST) was used to normalize the signals. Bands were quantified by ImageJ software.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!