NLRC5 Antibody

What is NLRC5 and why is it significant in immunological research?

NLRC5 belongs to the nucleotide-binding domain, leucine-rich repeat (NLR) family of cytosolic pattern recognition receptors that are central to health and disease processes. As one of the most enigmatic NLRs, NLRC5 is highly expressed in hematopoietic cells and has several critical functions that make it a valuable research target . NLRC5 regulates MHC class I gene expression, participates in NLRP3 inflammasome activation, and regulates inflammatory cell death (PANoptosis) . Polymorphisms in NLRC5 are associated with susceptibility to various infectious and inflammatory diseases as well as cancers, making it both a biomarker and potential therapeutic target . NLRC5 has the longest LRR domain of all human NLR members and is evolutionarily related to NOD1, NOD2, and NLRC3 .

What are the optimal tissue sources for studying NLRC5 expression?

NLRC5 exhibits differential expression patterns across tissues, with particularly high expression in immune cells. For initial characterization studies, researchers should consider:

• Spleen tissue, which shows robust NLRC5 expression that can be further enhanced with IFNγ treatment (10ng/ml for 16h)

• Leukocyte populations, as human leukocyte cDNA libraries have been successfully used for NLRC5 cloning

• Cancerous tissues, where NLRC5 expression may be altered compared to healthy counterparts

Researchers should be aware that NLRC5 expression levels can vary significantly between tissue types and can be upregulated under specific conditions, such as hemolytic diseases or after treatment with PAMPs like Pam3 .

How do I select the appropriate NLRC5 antibody for my research application?

When selecting an NLRC5 antibody, consider the following experimental factors:

• Target species: Ensure the antibody detects NLRC5 in your species of interest (human, mouse, rat)

• Application compatibility: Verify the antibody is validated for your specific application (Western blot, immunohistochemistry, ELISA, etc.)

• Epitope location: Different antibodies target different regions of NLRC5 (e.g., amino acids 117-136 or 1855-1866)

• Validation data: Review published literature or manufacturer data showing successful detection of endogenous NLRC5

For Western blotting applications, polyclonal antibodies like anti-NLRC5 (mouse) pAb (IN113) have been successfully used to detect endogenous mouse NLRC5 in splenocytes, particularly after IFNγ stimulation .

What is the optimal Western blot protocol for detecting endogenous NLRC5?

Detecting endogenous NLRC5 requires careful optimization due to its high molecular weight (approximately 204.6 kDa) . A successful Western blot protocol includes:

• Cell preparation: Use 5x10^6 splenocytes, with or without IFNγ stimulation (10ng/ml for 16h)

• Protein separation: Run samples on 8% SDS-PAGE under reducing conditions

• Transfer: Perform overnight transfer to nitrocellulose membrane to ensure complete transfer of high molecular weight NLRC5

• Antibody incubation: Use anti-NLRC5 antibody at appropriate dilution (e.g., 1:500 for anti-NLRC5 mouse pAb)

• Detection: Apply HRP-conjugated secondary antibody (e.g., 1:5000 dilution) and visualize using chemiluminescence

• Controls: Include actin antibody as a loading control

This protocol has been successful in detecting the upregulation of NLRC5 in response to IFNγ treatment in mouse splenocytes .

How can I induce and measure NLRC5 expression in experimental systems?

NLRC5 expression can be significantly modulated by various stimuli. Based on experimental evidence:

The upregulation of NLRC5 can be measured at both mRNA level (using RT-PCR with NLRC5-specific primers) and protein level (using Western blot with validated anti-NLRC5 antibodies) .

What controls should be included when performing immunoblotting with NLRC5 antibodies?

To ensure reliable and interpretable results when using NLRC5 antibodies for immunoblotting, include these essential controls:

• Positive control: IFNγ-stimulated cells known to express NLRC5 (e.g., splenocytes treated with 10ng/ml IFNγ for 16h)

• Negative control: Untreated cells with basal NLRC5 expression

• Loading control: Actin antibody to normalize protein loading across samples

• Specificity control: When possible, include NLRC5-deficient samples (Nlrc5^−/−) or cells treated with NLRC5-targeting siRNA

• Molecular weight marker: To confirm the ~200 kDa size of NLRC5

These controls help validate antibody specificity and ensure proper interpretation of bands observed at the expected molecular weight of NLRC5.

How can I use NLRC5 antibodies to study the PANoptosome and inflammatory cell death?

NLRC5 has been identified as a driver of PANoptosis (an inflammatory cell death pathway) in response to specific ligands. To study this process:

• Experimental model: Use primary bone marrow-derived macrophages (BMDMs) from wild-type and Nlrc5^−/− mice

• Stimulation protocols:

  • Heme plus LPS

  • Heme plus R848

  • Heme plus Pam3

  • Heme plus TNF

• Detection methods:

  • Cell death: Measure using appropriate cell death assays

  • NLRC5 expression: Monitor using Western blot with anti-NLRC5 antibodies

  • PANoptosome formation: Analyze NLRC5 interactions with NLRP12 and other components

This approach allows investigators to assess the role of NLRC5 in forming the PANoptosome complex and driving inflammatory cell death in response to various stimuli .

What are the key considerations when using NLRC5 antibodies to study cancer immunosurveillance?

NLRC5 plays a complex role in cancer, with both tumor-suppressive and tumor-promoting functions depending on the cancer type:

• In melanoma models:

  • NLRC5 expression enhances tumor immunogenicity

  • NLRC5 facilitates antigen processing and presentation

  • NLRC5-expressing tumor cells elicit CD8+ T cell-dependent antitumor immunity

• In endometrial carcinoma:

  • NLRC5 promotes tumor progression

  • NLRC5 regulates DNA mismatch repair (MMR) status

  • NLRC5 suppresses the NF-κB pathway in tumor cells

When designing experiments to study NLRC5 in cancer:

• Use immunohistochemistry with validated anti-NLRC5 antibodies to assess NLRC5 expression in tumor tissues

• Combine with MHC class I staining to correlate NLRC5 levels with antigen presentation capacity

• Include markers for immune infiltration (CD8+ T cells) to assess immunosurveillance

• Consider the specific cancer context, as NLRC5 may have divergent roles in different malignancies

How can I investigate NLRC5's role in regulating MHC class I expression?

NLRC5 is a key regulator of MHC class I gene expression, making it important for tumor immunosurveillance. To study this function:

• Experimental system: Use tumor cell lines (such as B16 melanoma) with stable NLRC5 expression

• Detection methods:

  • Flow cytometry to assess surface MHC-I expression (H-2D^b and H-2K^b in mouse cells)

  • RT-PCR to measure expression of MHC-I and antigen processing machinery genes

  • Functional T cell assays to assess antigen presentation capacity

• Key readouts:

  • Expression of MHC-I heavy chains

  • Expression of β2-microglobulin

  • Expression of antigen processing machinery components (TAP1, TAP2, PSMB8, PSMB9)

Research has shown that NLRC5 expression in B16 melanoma cells (B16-5) induces a subset of IFNγ-inducible MHC-I antigen-processing pathway genes and enhances surface expression of MHC-I molecules .

Why might NLRC5 detection fail despite successful protein extraction?

Detection failures for NLRC5 can occur for several technical reasons:

• Inefficient transfer of high molecular weight proteins: NLRC5 is approximately 204.6 kDa, requiring overnight transfer to ensure complete migration to the membrane

• Inadequate induction: Basal NLRC5 levels may be below detection limits; consider stimulating cells with IFNγ (10ng/ml for 16h) to enhance expression

• Inappropriate gel percentage: Use 8% SDS-PAGE gels to achieve proper separation of high molecular weight proteins

• Antibody specificity issues: Different antibodies target distinct epitopes of NLRC5; consider trying antibodies targeting different regions (e.g., amino acids 117-136 vs. 1855-1866)

• Protein degradation: NLRC5 may be susceptible to proteolytic degradation; ensure samples are properly preserved with protease inhibitors

If detection problems persist, consider using positive controls such as cells transfected with NLRC5 expression vectors to validate antibody functionality .

How should I interpret differences in NLRC5 expression patterns across different disease models?

NLRC5 expression varies considerably across different disease contexts, requiring careful interpretation:

• Hemolytic diseases:

  • NLRC5 expression is significantly upregulated in whole blood and CD71+ cells from malaria patients

  • Similar upregulation occurs in sickle cell disease monocytes

  • This suggests that hemolysis and circulating free heme are associated with increased NLRC5 expression

• Cancer:

  • In some cancers, reduced NLRC5 expression is linked to impaired CD8+ T-cell activation and poor patient prognosis

  • In endometrial carcinoma, NLRC5 promotes tumor progression by inducing mismatch repair gene deficiency

These apparently contradictory findings reflect the complex, context-dependent roles of NLRC5. When comparing expression data across models, researchers should consider:

• The specific disease pathology

• Cell types being examined

• Potential confounding factors (e.g., inflammation, tissue-specific regulation)

• Different detection methods (protein vs. mRNA levels)

What are the potential artifacts when using NLRC5 antibodies in immunofluorescence studies?

When performing immunofluorescence with NLRC5 antibodies, researchers should be aware of these potential artifacts:

• Nonspecific binding: Due to the large size and complex domain structure of NLRC5, antibodies may exhibit cross-reactivity with other NLR family members

• Background signal: Particularly in tissues with high autofluorescence (e.g., liver, brain)

• Epitope masking: NLRC5's interactions with multiple binding partners may obscure antibody binding sites

• Subcellular localization artifacts: NLRC5 can shuttle between cytoplasm and nucleus, and fixation methods may affect this localization pattern

To minimize these issues:

• Include appropriate negative controls (isotype control antibodies, NLRC5-deficient tissues)

• Optimize fixation and permeabilization protocols for nuclear proteins

• Use peptide competition assays to confirm antibody specificity

• Consider complementary approaches (e.g., in situ hybridization) to validate protein expression patterns

How can NLRC5 antibodies be used to investigate therapeutic opportunities in cancer?

NLRC5's dual role in cancer presents intriguing therapeutic possibilities:

• Enhancing tumor immunogenicity:

  • NLRC5 expression in B16 melanoma cells (B16-5) renders them immunogenic

  • These NLRC5-expressing tumor cells efficiently present melanoma antigens to CD8+ T cells

  • This suggests potential for NLRC5-based immunotherapeutic strategies

• Targeting NLRC5 in cancers where it promotes progression:

  • In endometrial carcinoma, NLRC5 induces mismatch repair gene deficiency

  • This dMMR status may sensitize tumors to immune checkpoint inhibitors

  • Monitoring NLRC5 expression may help predict response to immunotherapy

Researchers can use NLRC5 antibodies to stratify patient samples and monitor therapeutic responses in preclinical models. Immunohistochemical assessment of NLRC5 expression in tumor biopsies may provide valuable predictive information regarding immune checkpoint inhibitor efficacy.

What are the current limitations in NLRC5 antibody technology and how might they be addressed?

Despite advances in antibody technology, several limitations affect NLRC5 research:

• Limited epitope coverage: Most commercially available antibodies target specific epitopes, potentially missing important post-translational modifications or splice variants

• Cross-reactivity concerns: NLRC5's evolutionary relationship with other NLR family members (NOD1, NOD2, NLRC3) creates potential for cross-reactivity

• Species restrictions: Many NLRC5 antibodies are species-specific, limiting comparative studies across model organisms

• Functional antibodies: Most available antibodies are for detection only, not for functional studies (neutralization, activation)

Future technological advances may include:

• Development of monoclonal antibodies targeting conserved epitopes across species

• Generation of conformation-specific antibodies that distinguish active vs. inactive NLRC5

• Creation of antibody panels targeting different functional domains of NLRC5

• Nanobodies or single-chain antibodies for improved tissue penetration in imaging applications