NLRP2 Antibody

What is NLRP2 and what are its structural characteristics?

NLRP2 (NALP2) is a 121 kDa cytosolic member of the NLRP family of proteins. Human NLRP2 is 1062 amino acids in length and contains an N-terminal Pyrin domain (aa 9-90), followed by a Nacht region and nine leucine-rich repeats (LRRs, aa 467-1033). There are four alternate isoforms that show various deletions or substitutions. Unlike other NLRP proteins, no close rodent counterparts have been reported for NLRP2, making it somewhat unique in cross-species research contexts .

How can NLRP2 be detected in cellular samples?

NLRP2 can be detected using specific monoclonal or polyclonal antibodies through various techniques including Western blotting, immunohistochemistry, and immunofluorescence. For Western blot applications, NLRP2 typically appears as a band of approximately 130 kDa under reducing conditions. Commercial antibodies such as Mouse Anti-Human NLRP2/NALP2 Monoclonal Antibody have been validated for detecting NLRP2 in cell lines including A549 human lung carcinoma and LNCaP human prostate cancer cells .

Which cell types express NLRP2?

NLRP2 is expressed in several cell types including macrophages, astrocytes, proximal tubular epithelial cells (PTECs), and human umbilical vein endothelial cells (HUVECs). Interestingly, expression patterns vary significantly depending on pathological conditions. For instance, NLRP2 is highly expressed in cystinotic PTECs but almost undetectable in PTECs derived from healthy subjects. Additionally, NLRP2 expression increases significantly in spinal astrocytes during inflammatory pain conditions .

What is the recommended protocol for NLRP2 Western blotting?

For Western blot detection of NLRP2, researchers should:

• Lyse cells in RIPA buffer

• Measure protein concentration using BCA Protein assay

• Resolve 30 μg of protein extracts on 10% SDS-PAGE

• Transfer to nitrocellulose membranes

• Probe with specific NLRP2 antibody (e.g., NALP2, 137569 from Abcam)

• Develop using ECL system

This methodology has been validated to detect NLRP2 as a specific band at approximately 130 kDa under reducing conditions. For optimal results, use Immunoblot Buffer Group 2 and ensure proper protein denaturation .

How can immunoprecipitation be performed to study NLRP2 protein interactions?

For immunoprecipitation of NLRP2 and associated proteins:

• Lyse cells in RIPA buffer and measure protein concentration

• Pre-clear 1.5 mg of total proteins with 20 μl Dynabeads Protein A for 1 hour at 4°C

• Incubate pre-cleared lysates overnight with 1 μg of anti-NLRP2 antibody or Rabbit IgG control

• Add 20 μl Dynabeads Protein A and incubate for additional 2 hours

• Wash beads three times with PBS plus 0.1% Tween and once with PBS

• Elute immunoprecipitated proteins with Laemmli sample buffer

• Analyze by Western blot

This approach has successfully demonstrated NLRP2 interaction with IKKα, providing insight into the mechanism of NF-κB regulation by NLRP2 .

How should NLRP2 antibodies be stored and handled to maintain optimal activity?

For optimal antibody performance, follow these storage guidelines:

• Store at -20 to -70°C for up to 12 months from the date of receipt

• After reconstitution, store at 2 to 8°C under sterile conditions for up to 1 month

• For longer storage after reconstitution, maintain at -20 to -70°C under sterile conditions for up to 6 months

• Use a manual defrost freezer and avoid repeated freeze-thaw cycles

These conditions help maintain antibody stability and specificity, ensuring consistent experimental results .

How does NLRP2 affect inflammatory responses?

NLRP2's role in inflammation varies by cell type:

• In macrophages: NLRP2 suppresses TNF-alpha production in response to LPS via NF-κB, while promoting proIL-1 beta cleavage and release via procaspase-1.

• In PTECs: NLRP2 acts as a positive regulator of inflammation. Overexpression increases production of proinflammatory cytokines (IL-6, IL-8) and chemokines. This effect is NF-κB dependent, as treatment with NF-κB inhibitor (BAY 11-7082) reduces cytokine production.

• In spinal astrocytes: During inflammatory pain, NLRP2 inflammasome expression increases significantly. On post-injection day 3 of Complete Freund Adjuvant (CFA)-induced pain, the percentage of astrocytes expressing NLRP2 increases from 8.34±1.5% to 20.59±1.6%, correlating with increased IL-1β production .

What is known about NLRP2's role in apoptotic regulation?

NLRP2 exhibits antiapoptotic properties in certain cell types. In PTECs, NLRP2 overexpression results in a significantly lower apoptotic cell rate compared to control cells when challenged with proapoptotic stimuli such as Actinomycin-D and TNF-α. Conversely, silencing NLRP2 in cystinotic PTECs leads to a significant increase in apoptosis rate. This antiapoptotic effect is likely mediated through NF-κB activation, as NF-κB has a well-established role in suppressing apoptosis .

What RNA interference strategies are effective for studying NLRP2 function?

Two effective RNA interference approaches have been documented:

SMART pool siRNA approach:

• Transfect cells with a mixture of four chemically synthesized siRNAs against NLRP2 at a final concentration of 200 nM

• Use an on-target plus control pool as negative control

• Assess knockdown efficiency 72 hours post-transfection

• For cytokine release measurements, replace media 72 hours after transfection and collect 24 hours later

This approach achieved significant reduction in NLRP2 expression in cystinotic PTECs, resulting in decreased cytokine production and increased apoptosis sensitivity .

Alternative siRNA approach:
In HUVECs, NLRP2 knockdown using siRNA technology achieved approximately 54% reduction in mRNA expression and significant reduction in protein expression. This effectively inhibited cell proliferation, suggesting potential applications in anti-angiogenic therapy .

How can researchers measure NF-κB activity in relation to NLRP2 function?

To assess the impact of NLRP2 on NF-κB activity, researchers can employ the TransAm NF-κB family kit to measure p52, p50, p65, RelB, and c-Rel binding to DNA consensus sequences in nuclear lysates:

• Lyse cells with NE-PER kit to obtain nuclear pellets

• Lyse nuclei in Complete lysis Buffer and measure protein concentration with Bradford assay

• Load 50 μg of protein extracts on each well of the assay plate

• Incubate with primary and secondary antibodies

• Measure absorbance at 450 nm

This method successfully demonstrated that NLRP2 overexpression in PTECs upregulates p65 and p50 NF-κB DNA-binding activity, helping elucidate the mechanism by which NLRP2 enhances inflammatory responses .

What approaches are effective for studying NLRP2 inflammasome formation in neural tissues?

For examining NLRP2 inflammasome formation in neural tissues such as spinal astrocytes:

• Induce inflammatory pain using Complete Freund Adjuvant (CFA) injection

• Collect spinal cord sections at specific time points (e.g., day 3 post-injection)

• Perform co-immunostaining with:

  • Anti-NLRP2 antibody

  • Anti-GFAP antibody (astrocyte marker)

  • Nuclear stain (e.g., DAPI)

• Analyze co-localization using quantitative image analysis

This approach revealed that while baseline co-localization of NLRP2 with astrocytes was approximately 8.34±1.5%, this significantly increased to 20.59±1.6% at the peak of inflammatory pain, indicating upregulation of NLRP2 inflammasome in astrocytes during inflammatory conditions .

How can researchers address the contradictory findings regarding NLRP2's role in NF-κB signaling?

The apparently contradictory findings regarding NLRP2's role in NF-κB signaling (inhibitory in macrophages, stimulatory in PTECs) require careful experimental design:

• Cell-type specific controls: Always include appropriate controls for each cell type studied

• Comprehensive pathway analysis: Examine multiple points in the NF-κB pathway, including:

  • IKK complex activation

  • IκB phosphorylation and degradation

  • p65/p50 nuclear translocation

  • DNA binding activity

  • Transcriptional activity using reporter assays

• Context-dependent analysis: Evaluate NLRP2 function under different stimulation conditions (basal, TNF-α, LPS, etc.)

• Isoform-specific effects: Consider the possibility that different NLRP2 isoforms (four have been identified) may have distinct functions

• Protein interaction network: Identify cell-type specific binding partners using immunoprecipitation followed by mass spectrometry

This comprehensive approach may help reconcile the divergent observations and establish a unified model of NLRP2 function that accounts for cell-type specificity .

What are the challenges in studying NLRP2 across species?

A significant challenge in NLRP2 research is the absence of close rodent counterparts, making traditional mouse models potentially problematic for translational research. To address this limitation:

• Consider human cell-based systems or humanized mouse models

• Use genomic editing approaches (CRISPR/Cas9) to introduce human NLRP2 into mouse models

• Focus on conserved pathways downstream of NLRP2 rather than direct orthologs

• Employ comparative studies examining the functionally closest NLRP family members in rodents

• Validate findings in human primary cells and tissues whenever possible

These approaches may help bridge the gap between rodent models and human NLRP2 biology, enabling more reliable translational research .

What emerging therapeutic applications might target NLRP2?

Based on current research findings, several potential therapeutic applications targeting NLRP2 are emerging:

• Anti-inflammatory therapies: In conditions where NLRP2 enhances NF-κB activity (as in cystinotic PTECs), NLRP2 inhibition might reduce pathological inflammation.

• Anti-angiogenic cancer treatments: Evidence suggests NLRP2 knockdown inhibits HUVEC viability, indicating potential for anti-angiogenic cancer therapy. Gene therapy or monoclonal antibodies could selectively inhibit NLRP2 function in tumors to suppress tumor angiogenesis.

• Pain management: Given NLRP2's overexpression in spinal astrocytes during inflammatory pain, targeting NLRP2 inflammasomes might represent a novel approach for managing chronic inflammatory pain conditions.

• Anti-fibrotic treatments: Transcriptomic analyses revealed NLRP2 upregulates profibrotic mediators, suggesting potential applications in treating fibrotic kidney diseases.

• Cell survival modulation: NLRP2's antiapoptotic effects suggest it could be targeted in conditions requiring enhanced apoptotic responses, such as certain cancers .

Table 1: NLRP2 Expression in Different Cell Types and Conditions

Table 2: NLRP2 Effects on NF-κB-regulated Cytokines in PTEC