NLRC4 Antibody

What is NLRC4 and how does it function in inflammasome activation?

NLRC4 (NLR family CARD domain-containing protein 4, also known as IPAF) is a member of the NOD-like receptor family that forms part of the innate immune system's cytosolic sensing apparatus. It consists of three key domains: a caspase activation and recruitment domain (CARD), a nucleotide-binding oligomerization domain (NOD), and leucine-rich repeats (LRR) . Unlike other inflammasome proteins such as NLRP3 and AIM2, NLRC4 does not directly interact with pathogen-associated molecular patterns (PAMPs). Instead, it functions as a scaffolding protein activated by NAIP proteins that directly bind bacterial ligands like flagellin and type three secretion system (T3SS) components .

Upon activation, NLRC4 undergoes oligomerization forming a wheel-shaped structure, which enables the recruitment and activation of pro-caspase-1, leading to pyroptosis (a form of inflammatory cell death) and the proteolytic processing of pro-inflammatory cytokines IL-1β and IL-18 into their active forms . NLRC4 can either directly contact pro-caspase-1 via its CARD domain or utilize the adapter protein ASC, with each pathway yielding different functional outcomes .

What are the recommended methods for detecting NLRC4 expression in different cellular systems?

For effective detection of NLRC4 expression, researchers should consider:

• Western blot analysis: Using validated anti-NLRC4 antibodies (such as clone 6H9B13) with appropriate positive controls. Expected molecular weight is approximately 116 kDa .

• Immunofluorescence microscopy: Detecting cytoplasmic localization of NLRC4, with particular attention to puncta formation upon inflammasome activation.

• qRT-PCR: For mRNA expression analysis, particularly useful for examining transcriptional regulation.

• Flow cytometry: For quantitative analysis of NLRC4 expression across cell populations.

The choice of detection method should align with experimental objectives. For studying protein-protein interactions, co-immunoprecipitation approaches as described in the literature are recommended . When using newly acquired antibodies, validation across multiple detection platforms is essential to confirm specificity.

Which cell types are most suitable for studying NLRC4 function?

NLRC4 inflammasome biology has been primarily studied in:

• Myeloid cells: Including circulating monocytes and neutrophils, which express high levels of NLRC4 and show robust inflammasome responses .

• Bone marrow-derived macrophages (BMDMs): These represent an excellent model system, particularly from wild-type and NLRC4-deficient mice for comparison studies .

• Intestinal epithelial cells (IECs): Critical for understanding the specialized host defense role of NLRC4 in mucosal immunity against enteric pathogens like Salmonella .

• Dendritic cells: These cells express NLRC4 and contribute to inflammasome-dependent immune responses .

When selecting cell types, researchers should consider the biological context of their research question. For instance, studies focused on enteric infections should incorporate intestinal epithelial cells, while those focused on systemic inflammation may benefit from examining multiple myeloid cell populations.

How should experiments be designed to study NLRC4 inflammasome activation?

Robust experimental design for NLRC4 inflammasome activation studies should include:

• Appropriate stimuli selection:

  • Bacterial infection (e.g., Salmonella Typhimurium at MOI 10-50 for 2 hours)

  • Purified bacterial components (flagellin, PrgJ-like rod proteins) with transfection reagents

  • Intraperitoneal injection of Salmonella for in vivo models

• Essential readouts:

  • IL-1β and IL-18 secretion (ELISA)

  • Pyroptosis measurement (LDH release assay)

  • Caspase-1 activation (Western blot for cleaved caspase-1)

  • ASC oligomerization (ASC oligomerization assay)

• Critical controls:

  • NLRC4-deficient cells (Nlrc4-/- mice)

  • Uninfected controls

  • Non-activating bacterial mutants (flagellin or T3SS deficient)

The ASC oligomerization assay, in particular, provides crucial information about inflammasome assembly dynamics. This involves cell lysis in buffer containing 1% Triton X-100, centrifugation to obtain pellets, cross-linking with disuccinimidyl suberate, and Western blot analysis of the cross-linked pellets .

What are the optimal protocols for co-immunoprecipitation studies involving NLRC4?

For effective co-immunoprecipitation (co-IP) of NLRC4 with its interaction partners:

• Lysis conditions:

  • Cell lysis in RIPA buffer containing protease inhibitors

  • Centrifugation at 13,000 × g at 4°C for 10 min

  • Protein content measurement to ensure consistency

• Immunoprecipitation:

  • Addition of validated anti-NLRC4 antibody with overnight rotation at 4°C

  • Precipitation of immune complexes using protein A/G magnetic beads

  • Thorough washing (3 times) with 1× PBST containing 1% Triton X-100

  • Elution with 1× loading buffer and heating at 95°C for 5 min

• Analysis:

  • Western blot for co-precipitated proteins such as:

    • NAIPs (direct activators)

    • ASC (adaptor protein)

    • Caspase-1 (effector protease)

    • Other potential interaction partners (e.g., VDR as recently discovered)

This approach has been successfully used to identify novel interactors of NLRC4, including the vitamin D receptor (VDR), which enhances NLRC4 inflammasome activation .

How can phosphorylation status of NLRC4 be effectively monitored?

NLRC4 phosphorylation, particularly at serine 533, has been implicated in its activation, though recent research suggests this might be context-dependent . To monitor NLRC4 phosphorylation:

• Antibody-based detection:

  • Phospho-specific antibodies targeting S533 for Western blot

  • Comparison with total NLRC4 levels

• Phosphomimetic models:

  • Utilize S533D phosphomimetic or S533A non-phosphorylatable NLRC4 mutants

  • In vivo and in vitro comparisons of function

• Mass spectrometry:

  • For comprehensive phosphorylation site mapping

  • Quantification of phosphorylation stoichiometry at different sites

Recent evidence suggests that phosphorylation may not be universally required for NLRC4 function, as S533A mutants can still form functional inflammasomes in certain contexts . Researchers should design experiments that can detect both phosphorylation-dependent and independent mechanisms of activation.

How can researchers distinguish between direct and indirect effects on NLRC4 inflammasome activation?

Distinguishing direct from indirect effects requires sophisticated experimental approaches:

• Reconstitution systems:

  • Expression of NLRC4 components in heterologous systems

  • Step-wise assembly with purified components in cell-free systems

• Sequential activation analysis:

  • Temporal profiling of signaling events using time-course experiments

  • Pharmacological inhibitors at different time points to identify critical steps

• Domain-specific mutations:

  • Targeted mutations of NLRC4 interaction domains

  • Analysis of the NOD domain's ADP/ATP exchange during activation

• Controls for parallel pathways:

  • NLRP3 inhibitors/knockouts to exclude cross-activation

  • Combined genetic approaches (e.g., NLRC4/NLRP3 double knockouts)

Research has revealed that a single ligand-bound NAIP molecule is sufficient to propagate NLRC4 oligomerization, demonstrating the exquisite sensitivity of this system . Additionally, recent studies have questioned the previously proposed role of NLRP3 in NLRC4 activation, highlighting the importance of rigorous controls when studying these interconnected pathways .

What are the current challenges in studying NLRC4 inflammasomopathies?

NLRC4 inflammasomopathies represent a growing category of autoinflammatory diseases with substantial challenges for research:

• Diagnostic challenges:

  • Early recognition of telltale symptoms

  • Rapid diagnosis using serum IL-18 as a biomarker

  • Genetic sequencing for confirmation

• Phenotypic heterogeneity:

  • Disease spectrum ranges from cold urticaria to NOMID to AIFEC

  • Understanding genotype-phenotype correlations

• Research model limitations:

  • Development of appropriate mouse models that recapitulate human disease

  • Patient-derived cell systems for personalized studies

• Therapeutic research considerations:

  • Timing of anti-inflammatory interventions

  • Targeted approaches (IL-18BP, anti-IFNγ antibodies)

  • Assessment of experimental therapies

Researchers studying NLRC4 inflammasomopathies should design experiments that can capture both systemic inflammation and tissue-specific effects, particularly focusing on gastrointestinal manifestations that are prominent in conditions like AIFEC (autoinflammation with infantile enterocolitis).

How should researchers approach contradictory data regarding NLRC4 function?

The field of NLRC4 research has seen several areas of conflicting findings, particularly regarding phosphorylation requirements and interactions with other inflammasomes. To navigate these contradictions:

• Systematic comparison of experimental conditions:

  • Cell types used (primary vs. cell lines)

  • Activation stimuli and their purity

  • Genetic backgrounds of mouse models

• Multiple methodological approaches:

  • Combine biochemical, genetic, and imaging techniques

  • In vitro and in vivo validation of findings

• Careful genetic model design:

  • Use of cleanly generated knockouts rather than knockdowns

  • Targeted mutations (e.g., S533D phosphomimetic or S533A non-phosphorylatable NLRC4)

  • Validation across different genetic backgrounds

• Collaborative resolution efforts:

  • Direct comparison experiments between laboratories

  • Standardization of key protocols and reagents

Recent research has helped clarify some contradictions, such as demonstrating that NLRP3 is not essential for NLRC4 function, contrary to earlier proposals, and clarifying that phosphorylation at S533 may not be universally required for NLRC4 activation .

What are the common pitfalls in NLRC4 antibody-based experiments and how can they be avoided?

Researchers should be aware of several potential pitfalls:

• Antibody specificity issues:

  • Cross-reactivity with other NLR family members

  • Batch-to-batch variation in antibody performance

• Signal detection challenges:

  • Background in Western blot applications

  • Nonspecific binding in immunofluorescence

• Activation state specificity:

  • Many antibodies cannot distinguish between active and inactive NLRC4

  • Conformational epitopes may be affected by sample preparation

To mitigate these issues:

• Rigorous validation:

  • Use NLRC4-deficient cells as negative controls

  • Compare multiple antibody clones

  • Validate across different applications (WB, IF, IP)

• Optimized protocols:

  • Determine optimal antibody concentrations

  • Adjust blocking conditions to minimize background

  • Consider native vs. denaturing conditions for epitope accessibility

• Complementary approaches:

  • Combine antibody-based methods with genetic reporters

  • Use functional readouts alongside direct NLRC4 detection

How can researchers ensure reproducibility in NLRC4 inflammasome activation experiments?

Reproducibility challenges in NLRC4 research can be addressed through:

• Standardized activation protocols:

  • Consistent bacterial growth conditions for Salmonella (log phase cultures)

  • Defined MOI and infection duration

  • Controlled transfection conditions for bacterial components

• Comprehensive reporting:

  • Detailed methods sections including cell culture conditions

  • Specific reagent information including antibody clones and catalog numbers

  • Raw data availability

• Multiple readouts of activation:

  • Combine cytokine measurements with cell death assays

  • Assess both ASC-dependent and independent pathways

  • Monitor both IL-1β and IL-18 production

• Biological and technical replicates:

  • Minimum of three independent experiments

  • Multiple technical replicates within experiments

  • Use of different primary cell preparations

What quality control measures are essential for NLRC4 antibody validation?

Comprehensive quality control for NLRC4 antibodies should include:

• Specificity testing:

  • Western blot analysis using lysates from wild-type and NLRC4-deficient cells

  • Testing against recombinant NLRC4 protein

  • Cross-reactivity assessment with other NLR family members

• Functional validation:

  • Ability to detect NLRC4 in both resting and activated states

  • Effectiveness in co-immunoprecipitation applications

  • Performance in fixed tissue samples

• Application-specific optimization:

  • Titration for optimal signal-to-noise ratio

  • Buffer compatibility testing

  • Epitope accessibility in different applications

• Lot-to-lot consistency assessment:

  • Comparative testing between antibody lots

  • Retention of reference lots for benchmarking

  • Documentation of any performance variations

What are the emerging methodologies for studying NLRC4 dynamics and interactions?

The field is advancing with several promising methodologies:

• Advanced imaging techniques:

  • Super-resolution microscopy for inflammasome structure analysis

  • Live-cell imaging with fluorescently tagged NLRC4

  • Single-molecule tracking for activation dynamics

• Proteomic approaches:

  • Proximity labeling to identify transient interactors

  • Quantitative proteomics for global changes during activation

  • Cross-linking mass spectrometry for structural insights

• Single-cell techniques:

  • Single-cell RNA sequencing for heterogeneity in responses

  • Combined protein and RNA analysis at single-cell level

  • Spatial transcriptomics for tissue context

• Structural biology advances:

  • Cryo-electron microscopy of assembled inflammasomes

  • Hydrogen-deuterium exchange mass spectrometry for conformational changes

  • Solution NMR for dynamic structural alterations

How can researchers design experiments to investigate non-canonical functions of NLRC4?

Beyond inflammasome activation, NLRC4 has been implicated in other biological processes that warrant investigation:

• Tumor suppression:

  • Though recent research questions the role of NLRC4 in melanoma models , other cancer contexts should be explored

  • Design of conditional knockout models in relevant tissues

  • Analysis of NLRC4 expression and mutations in human tumor databases

• Tissue-specific functions:

  • Specialized roles in intestinal epithelial cells

  • Cell type-specific knockout models

  • Organoid systems for three-dimensional tissue context

• Inflammasome-independent signaling:

  • Separation of pyroptosis from cytokine processing

  • Investigation of nuclear translocation and potential transcriptional roles

  • Analysis of protein-protein interactions outside the inflammasome complex

• Environmental sensing:

  • Responses to metabolic signals

  • Interaction with vitamin D receptor and other nuclear receptors

  • Integration with other stress response pathways

When designing these experiments, researchers should consider both gain-of-function and loss-of-function approaches, as well as the potential for compensatory mechanisms through related inflammasome pathways.