il17rd Antibody

What is IL17RD and why is it important in immunological research?

IL17RD is a membrane protein belonging to the interleukin-17 receptor (IL-17R) family. It serves as a critical component of the interleukin-17 receptor signaling complex, interacting with IL-17R in a manner that does not require the presence of interleukin itself. The importance of IL17RD extends beyond IL-17 signaling, as it also affects fibroblast growth factor signaling, capable of either inhibiting or stimulating growth through MAPK/ERK signaling pathways. Additionally, recent research has revealed that IL17RD plays a crucial role in regulating Toll-like receptor (TLR) signaling, functioning as a negative regulator to prevent excessive inflammatory responses. This multifaceted role makes IL17RD a significant target for immunological research, particularly in understanding the cross-regulation between different receptor families in the immune system .

What applications are IL17RD antibodies suitable for?

Based on the available data, IL17RD antibodies have been validated for several research applications:

• Western Blot (WB): Useful for detecting IL17RD protein expression in cell lysates, with recommended dilutions ranging from 1:500-1:2000. Studies have successfully used WB to detect IL17RD in various cell lines including A172 and 231 cells .

• Immunohistochemistry (IHC): Effective for visualizing IL17RD expression in tissue sections, with optimal dilutions typically between 1:50-1:100. IL17RD has been successfully detected in paraffin-embedded tissues including human kidney and cervical cancer tissues .

• Flow Cytometry: Suitable for detecting IL17RD in cell populations such as the K562 human chronic myelogenous leukemia cell line, allowing researchers to quantify expression levels across different cell populations .

• Immunoprecipitation (IP): Valuable for studying protein-protein interactions, such as the association between IL17RD and other components of the receptor complex like IL-17R and TRAF6 .

• ELISA: Applicable for quantitative detection of IL17RD, with recommended dilutions of 1:5000-1:10000 .

How should I validate the specificity of an IL17RD antibody for my research?

Validating antibody specificity is critical for ensuring reliable experimental results. For IL17RD antibodies, a comprehensive validation approach should include:

• Positive and negative controls: Use cell lines known to express IL17RD (such as K562, A172, or 231 cells) as positive controls. Include appropriate negative controls, such as isotype control antibodies (e.g., Catalog # AB-108-C as demonstrated in flow cytometry experiments) to verify specific binding .

• Blocking experiments: Pre-incubate the antibody with recombinant IL17RD protein before application to demonstrate that binding can be specifically blocked.

• Multiple detection methods: Cross-validate antibody performance using different techniques (e.g., if using the antibody for IHC, confirm expression pattern using WB or flow cytometry).

• Knockdown/knockout validation: Compare antibody staining between wild-type cells and those where IL17RD expression has been reduced or eliminated through genetic approaches such as siRNA, shRNA, or CRISPR-Cas9.

• Epitope mapping: If possible, verify that the antibody recognizes the expected region of IL17RD (e.g., antibodies targeting the Ala27-Arg299 region as specified in product AF2275) .

What are the optimal storage and handling conditions for IL17RD antibodies?

To maintain antibody performance and extend shelf-life, follow these research-validated practices:

• Storage temperature: Store IL17RD antibodies at -20°C for long-term preservation, as specified in product documentation .

• Avoid freeze/thaw cycles: Repeated freezing and thawing can degrade antibody performance. Aliquot antibodies upon receipt to minimize freeze/thaw cycles .

• Proper dilution buffers: Use recommended buffers for dilution. For example, some IL17RD antibodies are provided in PBS with 0.05% NaN3 and 40% Glycerol at pH 7.4, which helps maintain stability .

• Centrifugation: Prior to opening a vial of antibody, briefly centrifuge to ensure complete recovery of contents, particularly after shipping or storage .

• Working solution stability: Once diluted, antibody solutions should be used promptly. If storage of diluted antibody is necessary, keep at 4°C for short periods (1-2 weeks) with appropriate preservatives.

How can I study the interaction between IL17RD and other components of the IL-17 receptor complex?

Investigating IL17RD interactions with other receptor components requires sophisticated approaches:

• Co-immunoprecipitation (Co-IP): This technique has successfully demonstrated that IL17RD associates with IL-17R and IL-17RB. The protocol involves:

  • Co-expressing IL17RD and IL-17R in HEK293T cells

  • Lysing cells in appropriate buffer conditions

  • Immunoprecipitating with anti-IL17RD or anti-IL-17R antibodies

  • Analyzing precipitated complexes by Western blot to detect interacting partners

• Domain mapping studies: Research has revealed that both full-length IL17RD and mutants lacking the extracellular domain (IL17RDΔECD) can interact with IL-17R, whereas mutants lacking the intracellular domain (IL17RDΔICD) retain the ability to interact but cannot mediate signaling. Generate similar domain constructs to map interaction interfaces .

• Proximity ligation assay (PLA): This technique can be employed to visualize protein-protein interactions in situ, providing spatial information about where IL17RD interacts with other receptor components within cells.

• FRET/BRET analyses: These biophysical approaches can measure real-time interactions between fluorescently or bioluminescently tagged IL17RD and other receptor components in living cells.

• Crosslinking mass spectrometry: For detailed mapping of interaction interfaces, chemical crosslinking followed by mass spectrometry can identify specific amino acid residues involved in protein-protein contacts .

What experimental approaches can be used to investigate IL17RD's role in regulating TLR signaling?

Recent research has established IL17RD as a negative regulator of TLR signaling. To investigate this function:

• Reporter gene assays: Luciferase reporter systems responsive to NF-κB activation can measure how IL17RD expression modulates TLR signaling. Research has shown that IL17RDΔICD acts as a dominant-negative regulator, inhibiting signaling in a dose-dependent manner .

• Knockout/knockdown studies: Compare TLR responses in wild-type versus IL17RD-deficient cells or animals. Published data indicates that Il17rd−/− mice display enhanced susceptibility to TLR-induced septic shock, demonstrating IL17RD's role as a negative regulator .

• Analysis of IL17RD-SEFIR domain: The intracellular SEFIR domain of IL17RD targets TIR adaptor proteins to inhibit TLR downstream signaling. Design experiments to test how mutations within this domain affect its regulatory function .

• Pro-inflammatory gene expression analysis: Measure expression of TLR-induced genes (e.g., cytokines/chemokines like IL-6, IL-8, CXCL1, CXCL2; cytoplasmic proteins like COX2; extracellular matrix proteins like Mmp3, Mmp9) in IL17RD-sufficient versus IL17RD-deficient conditions .

• Protein-protein interaction studies: Investigate how IL17RD interacts with components of the TLR signaling complex, particularly focusing on TIR domain-containing adaptor proteins .

How do I differentiate between various splice variants of IL17RD in my experiments?

IL17RD undergoes alternative splicing to generate multiple transcript variants encoding distinct isoforms. To differentiate between these variants:

• Isoform-specific antibodies: Where available, use antibodies raised against unique regions present in specific splice variants. Check epitope information in antibody documentation to determine which isoforms will be recognized .

• PCR-based approaches:

  • Design primer pairs that span exon-exon junctions unique to particular splice variants

  • Use quantitative RT-PCR with isoform-specific primers to quantify relative expression levels

  • Employ nested PCR strategies for low-abundance variants

• Western blot analysis: Different isoforms may have distinct molecular weights. The calculated molecular weight of full-length IL17RD is approximately 82 kDa, but observed weights may vary due to post-translational modifications and splice variations .

• Recombinant expression systems: Express individual splice variants in cell systems to generate standards for comparison with endogenous proteins.

• Mass spectrometry: For definitive identification, isolate IL17RD protein and perform mass spectrometry analysis to identify peptides unique to specific splice variants.

What are the critical controls when studying IL17RD-mediated signaling in cell culture?

Robust experimental design requires appropriate controls to ensure valid interpretation of results:

• Pathway stimulation controls:

  • For IL-17 signaling: Include untreated and IL-17-treated conditions (note that IL17RD-IL17R interaction appears to be constitutive and not enhanced by IL-17 treatment)

  • For TLR signaling: Include appropriate TLR ligands (e.g., LPS for TLR4) with and without IL17RD modulation

• Expression controls:

  • When overexpressing IL17RD or mutant constructs (e.g., IL17RDΔECD, IL17RDΔICD), verify expression levels by Western blot

  • For dose-dependent experiments, demonstrate correlation between IL17RD expression and functional outcomes

• Specificity controls:

  • Use IL17RD knockout or knockdown cells alongside wild-type cells

  • Include dominant-negative constructs like IL17RDΔICD, which has been shown to block IL-17RD-mediated signaling

• Downstream readouts:

  • Monitor multiple signaling outputs (e.g., NF-κB activation, MAPK phosphorylation)

  • Measure expression of known IL-17-responsive genes (cytokines, chemokines, matrix proteins)

• Cell type considerations:

  • Validate findings across multiple cell types as IL17RD function may vary (e.g., MEFs, NIH3T3, HEK293T have all been used in IL17RD research)

What are common challenges in detecting IL17RD by immunohistochemistry, and how can they be addressed?

Immunohistochemical detection of IL17RD can present several challenges:

• Tissue-specific expression patterns: IL17RD exhibits distinct localization patterns in different tissues. In human kidney, for example, specific labeling is localized to the cytoplasm of glomeruli and endothelial cells in capillaries in connective tissue . When establishing IHC protocols:

  • Begin with tissues known to express IL17RD (kidney, cervical tissue)

  • Optimize antibody dilution, starting with manufacturer recommendations (1:40-1:50)

  • Include appropriate positive and negative control tissues

• Antigen retrieval optimization:

  • For paraffin-embedded sections, test different antigen retrieval methods (heat-induced in citrate buffer pH 6.0 or EDTA buffer pH 9.0)

  • Adjust retrieval time and temperature based on signal-to-noise ratio

• Background reduction:

  • Block endogenous peroxidase activity (if using HRP-based detection)

  • Use appropriate serum blocking (e.g., if using goat anti-human IL17RD antibody, block with normal goat serum)

  • Consider tissue-specific autofluorescence quenching for immunofluorescence applications

• Signal amplification:

  • For low-abundance expression, consider using tyramide signal amplification

  • Validated systems like the Anti-Goat HRP-DAB Cell & Tissue Staining Kit have been successfully employed at concentrations of 15 μg/mL with overnight incubation at 4°C

• Counterstaining considerations:

  • Hematoxylin counterstaining provides context for IL17RD localization

  • For multi-color immunofluorescence, select compatible fluorophores with minimal spectral overlap

How can I optimize detection of IL17RD in flow cytometry experiments?

Flow cytometry allows quantitative analysis of IL17RD expression at the single-cell level. Optimization strategies include:

• Cell preparation:

  • Use gentle cell dissociation methods to preserve surface epitopes

  • For intracellular detection, ensure appropriate fixation and permeabilization

  • Maintain viability with proper buffers and temperature control

• Antibody titration:

  • Perform serial dilutions to determine optimal antibody concentration

  • K562 human chronic myelogenous leukemia cells provide a useful positive control system

• Fluorophore selection:

  • For direct conjugates, select bright fluorophores appropriate for your cytometer

  • For indirect detection, validated secondary antibodies like Phycoerythrin-conjugated Anti-Goat IgG have been successfully used

• Controls:

  • Include appropriate isotype controls (e.g., Catalog # AB-108-C has been validated)

  • Use unstained cells and single-color controls for compensation

  • Consider fluorescence-minus-one (FMO) controls for accurate gating

• Analysis considerations:

  • Gate on viable, single cells to eliminate artifacts

  • Present data as histogram overlays (filled histogram for IL17RD staining, open histogram for isotype control) to demonstrate specific staining

  • Quantify using appropriate metrics (median fluorescence intensity rather than mean for non-normal distributions)

How should I interpret conflicting data between different detection methods for IL17RD?

Discrepancies between detection methods are common in protein research and require systematic troubleshooting:

• Method-specific limitations:

  • Western blot detects denatured protein and may miss conformation-dependent epitopes

  • Flow cytometry accesses cell surface proteins more readily than intracellular targets

  • IHC provides spatial information but may be affected by fixation artifacts

• Antibody epitope considerations:

  • Different antibodies may recognize distinct epitopes or isoforms

  • Check if antibodies target the extracellular domain (ECD) or intracellular domain (ICD) of IL17RD, as these may yield different results

  • Research has shown that IL17RD forms complexes with other proteins, potentially masking epitopes in certain contexts

• Experimental validation approaches:

  • Use multiple antibodies targeting different regions of IL17RD

  • Combine protein and mRNA detection methods (e.g., Western blot with RT-PCR)

  • Include knockdown/knockout controls to confirm specificity

• Biological explanations for discrepancies:

  • Post-translational modifications may affect epitope recognition

  • IL17RD undergoes alternative splicing, generating multiple variants

  • Protein localization may change based on cellular activation state

• Reporting recommendations:

  • Acknowledge discrepancies transparently in research communications

  • Provide detailed methods for each technique to facilitate replication

  • Present quantitative data from multiple approaches when possible

What emerging techniques show promise for studying IL17RD function and interactions?

Several cutting-edge approaches offer new possibilities for IL17RD research:

• CRISPR-Cas9 genome editing:

  • Generate precise IL17RD domain mutations to study structure-function relationships

  • Create reporter knock-ins to monitor endogenous IL17RD expression and localization

  • Perform CRISPR screens to identify novel IL17RD interactors or regulators

• Single-cell analyses:

  • Apply single-cell RNA-seq to characterize IL17RD expression heterogeneity across cell populations

  • Combine with protein detection (CITE-seq) to correlate transcript and protein levels

  • Use spatial transcriptomics to map IL17RD expression in tissue contexts

• Advanced imaging approaches:

  • Super-resolution microscopy to visualize IL17RD distribution at the nanoscale

  • Live-cell imaging with tagged IL17RD to track receptor dynamics and clustering

  • Correlative light and electron microscopy to relate IL17RD localization to ultrastructural features

• Structural biology:

  • Cryo-EM to determine the structure of IL17RD in complex with IL-17R and other components

  • Hydrogen-deuterium exchange mass spectrometry to map conformational changes upon ligand binding or complex formation

• Systems biology:

  • Multi-omics approaches integrating transcriptomics, proteomics, and phosphoproteomics

  • Mathematical modeling of IL17RD's dual role in IL-17 and TLR signaling networks

How might research on IL17RD inform therapeutic strategies for inflammatory diseases?

IL17RD's role in regulating both IL-17 and TLR signaling positions it as a potential therapeutic target:

• Targeting IL17RD in hyperinflammatory conditions:

  • Research has shown that IL17RD negatively regulates TLR signaling, with Il17rd−/− mice exhibiting enhanced susceptibility to TLR-induced septic shock

  • Approaches mimicking IL17RD's inhibitory effect could potentially dampen excessive TLR-mediated inflammation

• Modulating IL-17 signaling specificity:

  • IL17RD forms heteromeric complexes with IL-17R and IL-17RB

  • Therapeutic strategies could potentially target specific receptor complexes to selectively modulate IL-17 family cytokine responses

• Exploiting domain-specific functions:

  • The intracellular domain (ICD) of IL17RD is essential for signaling function, while IL17RDΔICD acts as a dominant-negative inhibitor

  • Peptide or small molecule mimetics of functional domains could offer selective pathway modulation

• Biomarker potential:

  • Expression patterns of IL17RD in tissues could serve as diagnostic or prognostic indicators

  • Detection of IL17RD in accessible specimens (using antibody-based methods) might inform patient stratification for IL-17-targeted therapies

• Combination approaches:

  • Understanding IL17RD's role in cross-regulation between IL-17 and TLR pathways could inform combination therapies targeting multiple inflammatory pathways

What statistical approaches are appropriate for analyzing IL17RD expression data?

Analyzing IL17RD expression requires rigorous statistical methods:

• For Western blot quantification:

  • Normalize band intensity to loading controls (β-actin, GAPDH)

  • For comparing multiple conditions, use ANOVA followed by appropriate post-hoc tests

  • Present data as fold-change relative to control conditions with error bars representing standard deviation or standard error

• For flow cytometry data:

  • Report median fluorescence intensity rather than mean for non-normal distributions

  • For comparing IL17RD expression across multiple cell populations or treatment conditions, use appropriate parametric (t-test, ANOVA) or non-parametric (Mann-Whitney, Kruskal-Wallis) tests based on data distribution

  • Present representative histograms alongside quantitative analyses

• For gene expression analysis:

  • Use validated reference genes for RT-qPCR normalization

  • Apply appropriate multiple testing correction when analyzing expression across numerous conditions

  • Consider time-course analyses to capture dynamic changes in IL17RD expression

• For protein interaction studies:

  • Quantify co-immunoprecipitation results relative to input controls

  • For dose-dependent effects (e.g., increasing amounts of IL17RDΔICD leading to lower reporter activity), fit appropriate regression models

• For in vivo studies:

  • Power analyses should guide animal numbers

  • Use survival curve analysis (Kaplan-Meier) for septic shock models

  • Report individual data points alongside means to show distribution