Frozen sections (thickness, 10 μm) of mice were blocked with 5% normal goat serum in tris-buffered saline (TBS) for 30 min, followed by incubation with primary antibodies overnight at 4°C in TBS containing 5% goat serum and 0.1% Triton. Primary antibodies used included rabbit monoclonal NF-κB (1:50, Cell Signaling Technology, Danvers, MA, USA) and rabbit monoclonal NLRP3 (1:100, Cell Signaling Technology, Danvers, MA, USA). After washing with TBS, the sections were incubated with anti-Mouse IgG (H+L), F(ab')2 Fragment (Alexa Fluor®488 Conjugate) (1:1000, Cell Signaling Technology, Danvers, MA, USA), and anti-rabbit IgG (H+L), F(ab')2 Fragment (Alexa Fluor®488 Conjugate) (1:1000, Cell Signaling Technology, Danvers, MA, USA) in TBS containing 5% goat serum and 0.1% Triton at room temperature for 1 h. The immunostaining was analyzed by using a laser scanning confocal microscope (Nikon, A1, Shanghai, China).
Nlrp3
Manufactured by Cell Signaling Technology
Sourced in United States, China, United Kingdom
NLRP3 is a laboratory equipment product that functions as a sensor for detecting cellular stress and damage. It is involved in the activation of the inflammasome complex, which plays a role in the immune response. The NLRP3 product is used for research purposes to study cellular signaling pathways and immune system processes.
Automatically generated - may contain errors
Lab products found in correlation
Il 1β, by Cell Signaling Technology (48 mentions)
Caspase 1, by Cell Signaling Technology (40 mentions)
Gapdh, by Cell Signaling Technology (30 mentions)
β actin, by Cell Signaling Technology (28 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (20 mentions)
Cleaved caspase 1, by Cell Signaling Technology (13 mentions)
Caspase 1, by Santa Cruz Biotechnology (12 mentions)
Nf κb, by Cell Signaling Technology (10 mentions)
Nf κb p65, by Cell Signaling Technology (10 mentions)
Cleaved caspase 3, by Cell Signaling Technology (9 mentions)
Gsdmd, by Abcam (9 mentions)
Cleaved il 1β, by Cell Signaling Technology (9 mentions)
Pvdf membrane, by Merck Group (9 mentions)
Interleukin 6 (il 6), by Cell Signaling Technology (8 mentions)
P erk, by Cell Signaling Technology (8 mentions)
Caspase 1, by Abcam (7 mentions)
Caspase 3, by Cell Signaling Technology (7 mentions)
Il 18, by Cell Signaling Technology (7 mentions)
Txnip, by Cell Signaling Technology (6 mentions)
Gapdh, by Abcam (6 mentions)
184 protocols using nlrp3
1
Immunostaining of NF-κB and NLRP3 in Mice
2
Molecular Mechanisms of Anti-Inflammatory Effects
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Phorbol 12-myristate 13-acetate (PMA), lipopolysaccharide (LPS), monosodium urate (MSU), and colchicine were purchased from Sigma-Aldrich (St. Louis, MO, USA). Neoastilbin (purity ≥ 98%) was purchased from Sichuan Victory Biological Technology Co., Ltd. (Chengdu, China). Fetal bovine serum (FBS) was purchased from Gibco (Scoresby, Australia). Penicillin–streptomycin solution was purchased from HyClone (Logan, UT, USA). Cell Counting Kit-8 (CCK-8) was purchased from Biosharp Life Sciences (Beijing, China). Enzyme-linked immunosorbent assay (ELISA) kits for interleukin-1β (IL-1β), interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) were purchased from MEIMIAN (Shanghai, China). NLRP3, ASC, Caspase-1, NF-κB p-p65, NF-κB p65, p-IKKα, IKKα, p-IκBα, IκBα, β-actin and Histone H3 antibodies were purchased from Cell Signaling Technology (Boston, MA, USA).
3
Immunoblotting Analysis of Gal-3, TXNIP, NLRP3 Proteins
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The tissues and cells’ samples were solubilized in RIPA lysis at 4°C and the protein was quantified using BCA assay; 50 µg protein was separated with 10% sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS/PAGE), and transferred on to a polyvinylidene difluoride membrane. Membrane was blocked with 5% non-fat in TBST for 1 h at 37°C and incubated with Gal-3 (1:500, Cell Signaling Technology), TXNIP (1:500, Cell Signaling Technology), NLRP3 (1:500, Cell Signaling Technology) and GAPDH (1:2000, Cell Signaling Technology) at 4°C overnight. Membrane was washed with TBST and incubated with goat anti-rabbit monoclonal IgG (1:10000; Cell Signaling Technology) secondary antibodies at room temperature for 1 h. Membrane was visualized with an ECL kit (Pierce Chemical Co.) and analyzed by Image-Pro Plus 6.0 software.
4
Western Blot Analysis of Inflammatory Signaling
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Total proteins were obtained by homogenizing colon tissue or lysating cells in ice-cold RIPA buffer with protease inhibitor (Thermo Fisher Scientific, Rockford, IL, USA). The proteins were separated by 10% SDS-PAGE, then transferred onto PVDF membranes. After blocking with 5% w/v nonfat milk diluted in TBS-T buffer for 1 h, the membranes were incubated overnight with primary antibodies at 4 °C. NLRP3, ASC, CASP1, NF-κB p65 (p65), phospho-p65 (p-p65), inhibitor of NFkappa B-alpha (IκBα), p-IκBα, Protein kinase B (Akt), p-Akt, extracellular signal-regulated kinase (ERK), p-ERK, p38 MAPK (p38), p-p38, c-Jun N-terminal kinase (JNK), and p-JNK were purchased from Cell Signaling Technology (Boston, MA, USA). Cyclooxygenase-2 (COX-2), inducible nitric oxide synthase (iNOS), and GAPDH antibodies were purchased from Abcam (Cambridge, UK). The membranes were washed 3 times. Then, they were incubated with corresponding secondary antibodies for 1 h at RT. To quantify the immunoreactive bands, we used a Bio-Rad enhanced chemiluminescence system and Image J software to visualize the immunoreactive bands (Laboratories, San Francisco, CA, USA).
5
Western Blot Analysis of Inflammasome Activation
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PBMCs were lysed with 200 µL of radioimmunoprecipitation assay (RIPA) buffer (Thermo Scientific, USA) containing protease inhibitor (Roche, Germany). Then, 50 µg of total protein was loaded into 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) loading buffer, and the proteins were transferred to polyvinylidene difluoride (PVDF) membranes. After sealing with 5% skimmed milk at 37 °C for 120 min, the PVDF membranes were incubated at 4 °C overnight with the primary antibodies against capase-1 p10 (Abcam, 1:1,000), IL-1β p17 (R&D Systems, Minneapolis, MN, USA; 1:1,000), NLRP3 (Cell Signaling, 1:1,000), caspase-1 (Cell Signaling, 1:1,000), ASC (Cell Signaling, 1:1,000), p-p65 (Cell Signaling, 1:1,000), and p-IκB α (Cell Signaling, 1:1,000) or Lamin-B (Cell Signaling, 1:1,000). Following that, the membranes were incubated with goat anti-rabbit IgG horseradish peroxidase (HRP)-conjugated secondary antibodies at 37 °C for 60 min. An automatic digital gel image analysis system Bio-Rad CFX-96 (Bio-Rad, CA, USA) was employed to determine and analyze the band densities.
6
NLRP1 Localization in Cell Fractions
7
Oxidative Stress Signaling Pathways
8
Inhibition of RAGE-Mediated Inflammation
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Dulbecco’s modified Eagle medium and penicillin–streptomycin solution were purchased from GIBCO (Grand Island, NY, USA). Fetal bovine serum was provided by Hyclone (Logan, UT, USA). Monoclonal rabbit antibodies, including anti-p38 MAPK, anti-phospho-p38 MAPK, anti-ERK1/2, anti-phospho-ERK1/2, anti-JNK, anti-phospho-JNK, anti-p65, anti-phospho-p65, NLRP3, and cleaved caspase-1 were obtained from Cell Signaling Technology (Boston, MA, USA). Receptor for AGE (RAGE) monoclonal rabbit antibody was purchased from AbCam (Cambridge, MA, USA). HRP-marked anti-β-actin antibody was supplied by Biorad (San Diego, CA, USA). 4′-Methoxyresveratrol (3,5-dyhydroxy-4′-methoxylstilbene) was purchased from Great Forest Biomedical (Hangzhou, China). BSA was obtained from ABCONE (Shanghai, China). KI and acetic acid were obtained from Sangon Biotech (Shanghai, China). Methylglyoxal, 2’,7’-dichlorodihydrofluorescein diacetate and other reagents were purchased from Sigma (St. Louis, MO, USA).
9
Mitochondrial Dynamics and Inflammatory Signaling
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Total lung tissue proteins were homogenized with a RIPA lysis buffer (Beyotime Biotechnology, China), and protein concentrations were quantified by a BCA assay kit (Thermo Fisher Scientific, USA). 30 μg of proteins per lane was separated through 10–15% denaturing polyacrylamide gels and transferred to PVDF membranes. The membranes were blocked with 5% nonfat dry milk and incubated with the following primary antibodies: TRAV1 and TRPA1 (Novus Biologicals, USA), MFF, DRP1, MFN2, OPA1, phosphorylated (phospho) NF-κB P65, total NF-κB P65, NLRP3 (Cell Signaling Technology, Danvers, MA, USA), and caspase-1 (Abcam, USA) overnight at 4°C. Membranes were then incubated with an HRP-conjugated anti-rabbit secondary antibody (Cell Signaling Technology, USA) and then visualized by chemiluminescent detection.
10
Whole Cell Lysate Protein Analysis in HK-2 Cells
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Whole cell lysate was prepared from cultured HK-2 cells in RIPA buffer (Biyuntian Bioengineering Institute, China). Soluble proteins from the culture medium were prepared as described previously. After measuring the protein concentration using BCA kit (Biyuntian Bioengineering Institute), equal amounts of total proteins were separated on SDS-PAGE gel and transferred onto a polyvinylidene difluoride (PVDF) membrane. The target protein was detected with one of the following primary antibodies (all from Cell Signaling, MA, USA, unless otherwise indicated) at 4 °C overnight: NLRP3 (15101S; Cell Signaling, MA, USA), TLR4 (bs-20594R; Bioss Bioengineering Institute, China), cleaved caspase 3 (9664S; Cell Signaling), caspase-3 (9665S; Cell Signaling), BAX (5023S; Cell Signaling), NF-κB (bs-0465R; Bioss Bioengineering Institute), p–NF–κB (3033T; Cell Signaling), TXNIP (14715S; Cell Signaling), and ABCG2 (bs-0662R; Bioss Bioengineering Institute). After the incubation with horseradish peroxidase-conjugated secondary antibodies at room temperature for 2 h. The signal was developed using the ECL system (Biyuntian Bioengineering Institute) according to the manufacturer's instructions. The signal density was analyzed using Quantity One Software (Bio-Rad, CA, USA) and the relative protein level was calculated as the density ratio of the target protein to β-actin (4970T; Cell Signaling).
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