Product: NLRP3 Antibody
Catalog: DF7438
Description: Rabbit polyclonal antibody to NLRP3
Application: WB IHC IF/ICC
Reactivity: Human, Mouse, Rat
Prediction: Pig, Bovine, Horse, Sheep, Rabbit, Dog
Mol.Wt.: 80~120kDa; 118kD(Calculated).
Uniprot: Q96P20
RRID: AB_2839376

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Product Info

Source:
Rabbit
Application:
WB 1:500-1:2000, IHC 1:50-1:200, IF/ICC 1:100-1:500
*The optimal dilutions should be determined by the end user.
*Tips:

WB: For western blot detection of denatured protein samples. IHC: For immunohistochemical detection of paraffin sections (IHC-p) or frozen sections (IHC-f) of tissue samples. IF/ICC: For immunofluorescence detection of cell samples. ELISA(peptide): For ELISA detection of antigenic peptide.

Reactivity:
Human,Mouse,Rat
Prediction:
Pig(90%), Bovine(90%), Horse(90%), Sheep(90%), Rabbit(90%), Dog(89%)
Clonality:
Polyclonal
Specificity:
NLRP3 Antibody detects endogenous levels of total NLRP3.
RRID:
AB_2839376
Cite Format: Affinity Biosciences Cat# DF7438, RRID:AB_2839376.
Conjugate:
Unconjugated.
Purification:
The antiserum was purified by peptide affinity chromatography using SulfoLink™ Coupling Resin (Thermo Fisher Scientific).
Storage:
Rabbit IgG in phosphate buffered saline , pH 7.4, 150mM NaCl, 0.02% sodium azide and 50% glycerol. Store at -20 °C. Stable for 12 months from date of receipt.
Alias:

Fold/Unfold

AGTAVPRL; AII/AVP; Angiotensin/vasopressin receptor AII/AVP like; Angiotensin/vasopressin receptor AII/AVP-like; C1orf7; Caterpiller protein 1.1; CIAS 1; CIAS1; CLR1.1; Cold autoinflammatory syndrome 1; Cold autoinflammatory syndrome 1 protein; Cryopyrin; Familial cold autoinflammatory syndrome; FCAS; FCU; LRR and PYD domains-containing protein 3; Muckle-Wells syndrome; MWS; NACHT; NACHT LRR and PYD containing protein 3; NALP 3; NALP3; NALP3_HUMAN; NLR family pyrin domain containing 3; NLRP3; PYPAF 1; PYPAF1; PYRIN containing APAF1 like protein 1; PYRIN-containing APAF1-like protein 1;

Immunogens

Immunogen:
Uniprot:
Gene(ID):
Expression:
Q96P20 NLRP3_HUMAN:

Predominantly expressed in macrophages. Also expressed in dendritic cells, B- and T-cells (at protein level) (PubMed:11786556) (PubMed:17164409). Expressed in LPS-treated granulocytes, but not in resting cells (at protein level) (PubMed:17164409). Expression in monocytes is very weak (at protein level) (PubMed:17164409). Expressed in stratified non-keratinizing squamous epithelium, including oral, esophageal and ectocervical mucosa and in the Hassall's corpuscles in the thymus. Also, detected in the stratified epithelium covering the bladder and ureter (transitional mucosa) (at protein level) (PubMed:17164409). Expressed in lung epithelial cells (at protein level) (PubMed:23229815). Expressed in chondrocytes (PubMed:12032915). Expressed at low levels in resting osteoblasts (PubMed:17907925).

Description:
This gene encodes a pyrin-like protein containing a pyrin domain, a nucleotide-binding site (NBS) domain, and a leucine-rich repeat (LRR) motif. This protein interacts with the apoptosis-associated speck-like protein PYCARD/ASC, which contains a caspase recruitment domain, and is a member of the NALP3 inflammasome complex. This complex functions as an upstream activator of NF-kappaB signaling, and it plays a role in the regulation of inflammation, the immune response, and apoptosis. Mutations in this gene are associated with familial cold autoinflammatory syndrome (FCAS), Muckle-Wells syndrome (MWS), chronic infantile neurological cutaneous and articular (CINCA) syndrome, and neonatal-onset multisystem inflammatory disease (NOMID). Multiple alternatively spliced transcript variants encoding distinct isoforms have been identified for this gene. Alternative 5' UTR structures are suggested by available data; however, insufficient evidence is available to determine if all of the represented 5' UTR splice patterns are biologically valid.
Sequence:
MKMASTRCKLARYLEDLEDVDLKKFKMHLEDYPPQKGCIPLPRGQTEKADHVDLATLMIDFNGEEKAWAMAVWIFAAINRRDLYEKAKRDEPKWGSDNARVSNPTVICQEDSIEEEWMGLLEYLSRISICKMKKDYRKKYRKYVRSRFQCIEDRNARLGESVSLNKRYTRLRLIKEHRSQQEREQELLAIGKTKTCESPVSPIKMELLFDPDDEHSEPVHTVVFQGAAGIGKTILARKMMLDWASGTLYQDRFDYLFYIHCREVSLVTQRSLGDLIMSCCPDPNPPIHKIVRKPSRILFLMDGFDELQGAFDEHIGPLCTDWQKAERGDILLSSLIRKKLLPEASLLITTRPVALEKLQHLLDHPRHVEILGFSEAKRKEYFFKYFSDEAQARAAFSLIQENEVLFTMCFIPLVCWIVCTGLKQQMESGKSLAQTSKTTTAVYVFFLSSLLQPRGGSQEHGLCAHLWGLCSLAADGIWNQKILFEESDLRNHGLQKADVSAFLRMNLFQKEVDCEKFYSFIHMTFQEFFAAMYYLLEEEKEGRTNVPGSRLKLPSRDVTVLLENYGKFEKGYLIFVVRFLFGLVNQERTSYLEKKLSCKISQQIRLELLKWIEVKAKAKKLQIQPSQLELFYCLYEMQEEDFVQRAMDYFPKIEINLSTRMDHMVSSFCIENCHRVESLSLGFLHNMPKEEEEEEKEGRHLDMVQCVLPSSSHAACSHGLVNSHLTSSFCRGLFSVLSTSQSLTELDLSDNSLGDPGMRVLCETLQHPGCNIRRLWLGRCGLSHECCFDISLVLSSNQKLVELDLSDNALGDFGIRLLCVGLKHLLCNLKKLWLVSCCLTSACCQDLASVLSTSHSLTRLYVGENALGDSGVAILCEKAKNPQCNLQKLGLVNSGLTSVCCSALSSVLSTNQNLTHLYLRGNTLGDKGIKLLCEGLLHPDCKLQVLELDNCNLTSHCCWDLSTLLTSSQSLRKLSLGNNDLGDLGVMMFCEVLKQQSCLLQNLGLSEMYFNYETKSALETLQEEKPELTVVFEPSW

Predictions

Predictions:

Score>80(red) has high confidence and is suggested to be used for WB detection. *The prediction model is mainly based on the alignment of immunogen sequences, the results are for reference only, not as the basis of quality assurance.

Species
Results
Score
Pig
90
Horse
90
Bovine
90
Sheep
90
Rabbit
90
Dog
89
Xenopus
0
Zebrafish
0
Chicken
0
Model Confidence:
High(score>80) Medium(80>score>50) Low(score<50) No confidence

PTMs - Q96P20 As Substrate

Site PTM Type Enzyme
Ubiquitination
S5 Phosphorylation
Y13 Phosphorylation
S161 Phosphorylation
S163 Phosphorylation
S198 Phosphorylation
T233 Phosphorylation
S295 Phosphorylation
S334 Phosphorylation
S387 Phosphorylation
S436 Phosphorylation
K496 Ubiquitination
S728 Phosphorylation
Y861 Phosphorylation
S975 Phosphorylation

Research Backgrounds

Function:

As the sensor component of the NLRP3 inflammasome, plays a crucial role in innate immunity and inflammation. In response to pathogens and other damage-associated signals, initiates the formation of the inflammasome polymeric complex, made of NLRP3, PYCARD and CASP1 (and possibly CASP4 and CASP5). Recruitment of proCASP1 to the inflammasome promotes its activation and CASP1-catalyzed IL1B and IL18 maturation and secretion in the extracellular milieu. Activation of NLRP3 inflammasome is also required for HMGB1 secretion. The active cytokines and HMGB1 stimulate inflammatory responses. Inflammasomes can also induce pyroptosis, an inflammatory form of programmed cell death. Under resting conditions, NLRP3 is autoinhibited. NLRP3 activation stimuli include extracellular ATP, reactive oxygen species, K(+) efflux, crystals of monosodium urate or cholesterol, amyloid-beta fibers, environmental or industrial particles and nanoparticles, cytosolic dsRNA, etc. However, it is unclear what constitutes the direct NLRP3 activator. Activation in presence of cytosolic dsRNA is mediated by DHX33. Independently of inflammasome activation, regulates the differentiation of T helper 2 (Th2) cells and has a role in Th2 cell-dependent asthma and tumor growth (By similarity). During Th2 differentiation, required for optimal IRF4 binding to IL4 promoter and for IRF4-dependent IL4 transcription. Binds to the consensus DNA sequence 5'-GRRGGNRGAG-3'. May also participate in the transcription of IL5, IL13, GATA3, CCR3, CCR4 and MAF (By similarity).

PTMs:

The disulfide bond in the pyrin domain might play a role in reactive oxygen species-mediated activation.

Ubiquitinated; undergoes both 'Lys-48'- and 'Lys-63'-linked polyubiquitination. Ubiquitination does not lead to degradation, but inhibits inflammasome activation (By similarity). Deubiquitination is catalyzed by BRCC3 and associated with NLRP3 activation and inflammasome assembly. This process can be induced by the activation of Toll-like receptors (by LPS), through a non-transcriptional pathway dependent on the mitochondrial production of reactive oxygen species, and by ATP.

Subcellular Location:

Cytoplasm>Cytosol. Inflammasome. Endoplasmic reticulum. Secreted. Nucleus.
Note: In macrophages, under resting conditions, mainly located in the cytosol, on the endoplasmic reticulum. After stimulation with inducers of the NLRP3 inflammasome, mitochondria redistribute in the vicinity of the endoplasmic reticulum in the perinuclear region, which results in colocalization of NLRP3 on the endoplasmic reticulum and PYCARD on mitochondria, allowing the activation of inflammasome assembly. After the induction of pyroptosis, inflammasome specks are released into the extracellular space where they can further promote IL1B processing and where they can be engulfed by macrophages. Phagocytosis induces lysosomal damage and inflammasome activation in the recipient cells (PubMed:24952504). In the Th2 subset of CD4(+) helper T-cells, mainly located in the nucleus. Nuclear localization depends upon KPNA2. In the Th1 subset of CD4(+) helper T-cells, mainly cytoplasmic (By similarity).

Golgi apparatus membrane.
Note: (Microbial infection) Upon HRSV infection, the protein is mainly located in lipid rafts in the Golgi membrane.

Extracellular region or secreted Cytosol Plasma membrane Cytoskeleton Lysosome Endosome Peroxisome ER Golgi apparatus Nucleus Mitochondrion Manual annotation Automatic computational assertionSubcellular location
Tissue Specificity:

Predominantly expressed in macrophages. Also expressed in dendritic cells, B- and T-cells (at protein level). Expressed in LPS-treated granulocytes, but not in resting cells (at protein level). Expression in monocytes is very weak (at protein level). Expressed in stratified non-keratinizing squamous epithelium, including oral, esophageal and ectocervical mucosa and in the Hassall's corpuscles in the thymus. Also, detected in the stratified epithelium covering the bladder and ureter (transitional mucosa) (at protein level). Expressed in lung epithelial cells (at protein level). Expressed in chondrocytes. Expressed at low levels in resting osteoblasts.

Subunit Structure:

Sensor component of NLRP3 inflammasomes. Inflammasomes are supramolecular complexes that assemble in the cytosol in response to pathogens and other damage-associated signals and play critical roles in innate immunity and inflammation. The core of NLRP3 inflammasomes consists of a signal sensor component (NLRP3), an adapter (ASC/PYCARD), which recruits an effector proinflammatory caspase (CASP1 and, possibly, CASP4 and CASP5). Within the complex, NLRP3 and PYCARD interact via their respective DAPIN/pyrin domains. This interaction initiates speck formation (nucleation) which greatly enhances further addition of soluble PYCARD molecules to the speck in a prion-like polymerization process. NLRP3 localizes at the end of each PYCARD filament. Clustered PYCARD nucleates the formation of CASP1 filaments through the interaction of their respective CARD domains, acting as a platform for CASP1 polymerization. CASP1 filament formation increases local enzyme concentration, resulting in trans-autocleavage and activation. Active CASP1 then processes IL1B and IL18 precursors, leading to the release of mature cytokines in the extracellular milieu and inflammatory response. Reconstituted ternary inflammasomes show star-shaped structures, in which multiple filaments, containing CASP1, protrude radially from a single central hub, containing the sensor protein and PYCARD. In this complex, the sensor protein is sub-stoichiometric to PYCARD, and PYCARD is further substoichiometric to CASP1, suggesting amplifications of signal transduction from the sensor, via the adapter, to the effector. Interacts with MEFV; this interaction targets NLRP3 to degradation by autophagy, hence preventing excessive IL1B- and IL18-mediated inflammation. Interacts with GBP5 (via DAPIN domain); this interaction promotes inflammasome assembly in response to microbial and soluble, but not crystalline, agents. Interacts with EIF2AK2/PKR; this interaction requires EIF2AK2 activity, is accompanied by EIF2AK2 autophosphorylation and promotes inflammasome assembly in response to specific stimuli. Interacts with PML (isoform PML-1) (via the leucine-rich repeat (LRR) domain); PML-mediated increase in NLRP3 inflammasome activation does not depend upon this interaction. Directly interacts with IRF4 (via the LRR domain); this interaction is required for optimal IRF4 binding to IL4 promoter and efficient IL4 transactivation during differentiation of Th2 helper T-cells (By similarity). Interacts (via NACHT domain) with DHX33 (via DEAH box). Interacts with PYDC5.

Family&Domains:

The pyrin domain (also called DAPIN domain or PYD) is involved in PYCARD-binding.

The LRR domain mediates the interaction with IRF4 and PML.

Intramolecular interactions between NACHT and leucine-rich repeat (LRR) domains may be important for autoinhibition in the absence of activating signal.

Belongs to the NLRP family.

Research Fields

· Cellular Processes > Cell growth and death > Necroptosis.   (View pathway)

· Human Diseases > Infectious diseases: Bacterial > Pertussis.

· Human Diseases > Infectious diseases: Viral > Influenza A.

· Organismal Systems > Immune system > NOD-like receptor signaling pathway.   (View pathway)

References

1). Activation of NLRP3 in microglia exacerbates diesel exhaust particles-induced impairment in learning and memory in mice. ENVIRONMENT INTERNATIONAL (PubMed: 31999974) [IF=11.8]

Application: WB    Species: mouse    Sample: BV2 cells

Fig. 5. Reactive oxygen species (ROS) generation, inflammatory markers, and metabolomics analyses of diesel exhaust particles (DEPs)-treated BV2 microglia in vitro. C: Protein levels of molecules involved in the NLRP3 pathway increased following DEPs treatment compared with those in control cells. One-way ANOVA followed by Turkey’s test, n = 3 biological replicates/group.

2). Water-extracted Lonicera japonica polysaccharide attenuates allergic rhinitis by regulating NLRP3-IL-17 signaling axis. Carbohydrate Polymers (PubMed: 36184153) [IF=11.2]

3). Investigation of pulmonary toxicity evaluation on mice exposed to polystyrene nanoplastics: The potential protective role of the antioxidant N-acetylcysteine. Science of The Total Environment (PubMed: 36155047) [IF=9.8]

4). GSK872 and necrostatin-1 protect retinal ganglion cells against necroptosis through inhibition of RIP1/RIP3/MLKL pathway in glutamate-induced retinal excitotoxic model of glaucoma. Journal of Neuroinflammation (PubMed: 36289519) [IF=9.3]

Application: WB    Species: Rat    Sample: R28 cells

Fig. 5 Activation of NLRP3 inflammasome triggered by glutamate excitotoxicity. a Protein levels of NLRP3 and NLRP3-related proteins in R28 cells after glutamate treatment. b Protein levels of NLRP3 and NLRP3-related proteins in the mouse retina after NMDA injury. c Representative immunofluorescence microphotographs of retinal sections stained with RBPMS (green), NLRP3 (red), and DAPI (blue) post-NMDA injection. Scale bar = 50 μm. GCL: ganglion cell layer; INL: inner nuclear layer; ONL: outer nuclear layer; CTL: the control group. The results were recorded as mean ± SD from at least three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 versus control or saline group. ns: not significant

Application: IF/ICC    Species: Rat    Sample: R28 cells

Fig. 5 Activation of NLRP3 inflammasome triggered by glutamate excitotoxicity. a Protein levels of NLRP3 and NLRP3-related proteins in R28 cells after glutamate treatment. b Protein levels of NLRP3 and NLRP3-related proteins in the mouse retina after NMDA injury. c Representative immunofluorescence microphotographs of retinal sections stained with RBPMS (green), NLRP3 (red), and DAPI (blue) post-NMDA injection. Scale bar = 50 μm. GCL: ganglion cell layer; INL: inner nuclear layer; ONL: outer nuclear layer; CTL: the control group. The results were recorded as mean ± SD from at least three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 versus control or saline group. ns: not significant

5). The role of NLRP3 in lead-induced neuroinflammation and possible underlying mechanism. Environmental Pollution (PubMed: 34182382) [IF=8.9]

Application: IF/ICC    Species: Rat    Sample: microglia

Fig. 3. Minocycline intervention reversed Pb induced NLRP3 activation. (A) Hippocampus slices were stained for NLRP3 (brown) and hematoxylin (blue) (40 × ), and arrows showed high definition (100 × ) of NLRP3 staining. (B) Co-localization of NLRP3 with microglia, staining with Iba-1 (red) and NLRP3 (green), and arrows showed the co-localization region (yellow) in microglia. (C) Number of Iba-1 and NLRP3 double-positive cells in DG region. The data are expressed as means ± SEM. *p < 0.05 vs. Con group, #p < 0.05 vs. Pb group. (D) Protein expression of NLRP3 and its molecules. (E) and (F) Gray analysis of protein expression. (G) mRNA level of NLRP3 in hippocampus tissues. Data are expressed as means ± SEM. *p < 0.05 vs. Con group, **p < 0.01 vs. Con group, #p < 0.05 vs. Pb group. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Application: WB    Species: Rat    Sample: microglia

Fig. 3. Minocycline intervention reversed Pb induced NLRP3 activation. (A) Hippocampus slices were stained for NLRP3 (brown) and hematoxylin (blue) (40 × ), and arrows showed high definition (100 × ) of NLRP3 staining. (B) Co-localization of NLRP3 with microglia, staining with Iba-1 (red) and NLRP3 (green), and arrows showed the co-localization region (yellow) in microglia. (C) Number of Iba-1 and NLRP3 double-positive cells in DG region. The data are expressed as means ± SEM. *p < 0.05 vs. Con group, #p < 0.05 vs. Pb group. (D) Protein expression of NLRP3 and its molecules. (E) and (F) Gray analysis of protein expression. (G) mRNA level of NLRP3 in hippocampus tissues. Data are expressed as means ± SEM. *p < 0.05 vs. Con group, **p < 0.01 vs. Con group, #p < 0.05 vs. Pb group. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

6). Combination of resolvin E1 and lipoxin A4 promotes the resolution of pulpitis by inhibiting NF-κB activation through upregulating sirtuin 7 in dental pulp fibroblasts. CELL PROLIFERATION (PubMed: 35411569) [IF=8.5]

Application: WB    Species: rat    Sample: Dental pulp fibroblasts

FIGURE 1 |Effects of combined administration of RvE1 and LXA4 on pro-inflammatory factor expression.(C) NLRP3, (D) caspase-1, (E) IL-1β and (F) IL-18 mRNA levels on LPS-induced DPFs detected by qPCR and (G) their protein levels tested by western blotting (normalized to that of β-tubulin).

7). Cardioprotective activity of ethyl acetate extract of Cinnamomi Ramulus against myocardial ischemia/reperfusion injury in rats via inhibiting NLRP3 inflammasome activation and pyroptosis. Phytomedicine (PubMed: 34673348) [IF=7.9]

8). Cinnamic acid preserves against myocardial ischemia/reperfusion injury via suppression of NLRP3/Caspase-1/GSDMD signaling pathway. Phytomedicine (PubMed: 35320770) [IF=7.9]

9). Formononetin protects against inflammation associated with cerebral ischemia-reperfusion injury in rats by targeting the JAK2/STAT3 signaling pathway. BIOMEDICINE & PHARMACOTHERAPY (PubMed: 35339827) [IF=7.5]

10). The gut microbiota attenuate neuroinflammation in manganese exposure by inhibiting cerebral NLRP3 inflammasome. BIOMEDICINE & PHARMACOTHERAPY (PubMed: 32768944) [IF=7.5]

Application: WB    Species: Rat    Sample: brain tissue

Fig. 4. Effect of Mn exposure and FMT on the brain-associated profile of NLRP3 inflammasome and inflammatory factors. (a–e) The activation level of NLRP3 inflammasome and inflammatory factors in the brain of rats. After FMT from healthy rats, the expression of IL-1β, IL-18, NLRP3, and TLR4 in brain was significantly downregulated, which were significantly different from that in Mn-treated group. *P < 0.05, **P < 0.01, ***P < 0.001.

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