Il31ra Antibody

What is IL31RA and why is it a significant research target?

IL31RA (Interleukin-31 Receptor A) is a ~100 kDa type I transmembrane glycoprotein classified as a type I cytokine receptor . It forms a heterodimeric complex with oncostatin M receptor (OSMR) to create the functional IL-31 receptor . This receptor is significant because:

• It mediates IL-31 signaling, activating STAT3 and to a lesser extent STAT1 and STAT5

• It plays a crucial role in the neuroimmune communication underlying pruritus (itching)

• It's implicated in multiple pathological conditions including atopic dermatitis, allergic asthma, and certain cancers

• It represents a therapeutic target for inflammatory and pruritic disorders

The 732 amino acid IL31RA contains a 19 aa signal sequence, a 500 aa extracellular domain, a 21 aa transmembrane domain, and a 192 aa cytoplasmic domain .

What are the primary applications for IL31RA antibodies in research?

IL31RA antibodies are valuable tools for numerous research applications:

• Western blotting for protein expression analysis

• Immunohistochemistry (IHC-P) for tissue localization studies

• Flow cytometry for cellular expression analysis

• Immunofluorescence for subcellular localization

• Enzyme-linked immunosorbent assays (ELISA)

• Functional blocking studies to investigate IL-31 signaling

Each application requires optimization of antibody concentration and conditions for specific experimental systems .

How do I select the appropriate IL31RA antibody for my specific experiment?

Selection criteria should include:

• Target epitope: Determine if you need antibodies targeting the extracellular domain (aa 20-516 is commonly used) or other regions

• Antibody type: Consider whether polyclonal (broader epitope recognition) or monoclonal (higher specificity) is appropriate

• Species reactivity: Verify reactivity with your species of interest (human IL31RA has 60%, 58%, 73%, and 70% amino acid identity with mouse, rat, canine, and bovine IL31RA ECD, respectively)

• Validated applications: Confirm the antibody has been validated for your specific application

• Clone information: For monoclonals, note the clone number (e.g., Clone #313308)

• Cross-reactivity: Check for known cross-reactions (e.g., approximately 5% cross-reactivity with recombinant mouse IL-31RA)

What cell types and tissues express IL31RA that I can use as positive controls?

Based on the literature, the following can serve as positive controls:

Cell Lines:

• U937 human histiocytic lymphoma cells

• K562 human chronic myelogenous leukemia cells

• THP-1 human acute monocytic leukemia cells

• MDA-MB-231 cells (basal-like breast cancer)

Primary Cells:

• Human PBMCs (especially after IFN-gamma treatment)

• CD14+ and CD56+ blood cells

• Macrophages

• Dorsal root ganglia neurons

• Keratinocytes

Tissues:

• Skin (particularly from atopic dermatitis patients)

• Dorsal root ganglia

• Lung epithelium (variable expression)

How should I optimize IL31RA antibody concentrations for different applications?

Western Blot:

• Start with 1:1000 dilution for polyclonal antibodies

• For monoclonal antibodies, begin with 0.1-1.0 μg/mL

• Include positive controls like THP-1 or U937 cells

• Expected band: ~100 kDa (may vary based on glycosylation and isoform)

Immunohistochemistry/Immunofluorescence:

• Use 5-15 μg/mL for initial testing

• For tissue sections, optimize antigen retrieval methods

• For cells, test both 4% paraformaldehyde and methanol fixation

• Counterstain with DAPI to visualize nuclei

Flow Cytometry:

• Start with 0.25-1.0 μg per 10^6 cells

• Include appropriate isotype controls (e.g., Rat IgG1)

• Pre-treat PBMCs with IFN-gamma (50 ng/mL for 20 hours) to upregulate expression for positive controls

Always perform antibody titration experiments to determine optimal concentration for your specific system .

What are the recommended sample preparation protocols for detecting IL31RA in different experimental systems?

Cell Lysate Preparation for Western Blot:

• Harvest cells at 70-80% confluence

• Lyse cells in RIPA buffer containing protease inhibitors

• For enhanced detection, consider pre-treating cells with IFN-gamma and bacterial lipopolysaccharides to upregulate IL31RA expression

• Include phosphatase inhibitors if analyzing phosphorylated forms

Tissue Preparation for IHC:

• Fix tissues in 10% neutral buffered formalin

• Process and embed in paraffin

• Cut 5 μm sections

• Perform antigen retrieval (citrate buffer pH 6.0 recommended)

• Block endogenous peroxidase activity and non-specific binding

Cell Preparation for Flow Cytometry:

• For adherent cells, detach using enzyme-free dissociation buffer

• For all cells, wash in PBS containing 1% BSA

• Fix if necessary (note that some epitopes may be sensitive to fixation)

• For enhanced detection in immune cells, stimulate with 50 ng/mL recombinant human IFN-gamma for 20 hours

How can I differentiate between the long and short isoforms of IL31RA in my experiments?

The detection of different IL31RA isoforms requires careful consideration:

Western Blot Approach:

• Use gradient gels (4-15%) to achieve better separation of different molecular weight isoforms

• The long form (~745 aa) and the 732 aa form will appear at approximately 100 kDa

• The short form (~560 aa) will appear at approximately 70-80 kDa

• Use positive controls expressing known isoforms

PCR-Based Approach:

• Design primers that span regions specific to each isoform

• The long form (signaling) recruits STAT3, 5, or 1

• The short form (inhibitory) does not recruit STATs and inhibits IL-31 signaling

• qRT-PCR can be used to quantify the relative expression of each isoform

Functional Characterization:

• The long form activates STAT3/5/1 signaling pathways

• The short form has inhibitory functions

• Assess downstream signaling events (STAT phosphorylation) to differentiate functional consequences of the isoforms

The ratio of these forms and their co-expression with OSMR determines a cell's response to IL-31 .

How can I use IL31RA antibodies to investigate neurogenic inflammation and pruritus mechanisms?

IL31RA plays a crucial role in neuroimmune communication, particularly in pruritus:

Methodological Approach:

• Co-localization studies: Use IL31RA antibodies in combination with neuronal markers (TRPV1, TRPA1) in dorsal root ganglia samples to identify sensory neurons expressing IL31RA

• Calcium imaging:

  • Isolate dorsal root ganglia neurons

  • Load with calcium-sensitive dyes

  • Apply IL-31 and measure calcium influx

  • Block with IL31RA antibodies to confirm specificity

• Electrophysiology:

  • Perform patch-clamp recordings on IL31RA+ neurons

  • Measure changes in membrane potential after IL-31 stimulation

  • Use IL31RA blocking antibodies to confirm receptor specificity

• In vivo models:

  • Administer IL31RA blocking antibodies in mouse models of atopic dermatitis

  • Quantify scratching behavior before and after treatment

  • Assess skin inflammation histologically

A detailed understanding of IL31RA in sensory neurons has revealed that, beyond triggering pruritus, IL-31 can also activate a neurogenic anti-inflammatory pathway involving calcitonin gene-related peptide (CGRP) release, which suppresses type 2 inflammation in certain contexts .

What strategies can be employed to investigate the crosstalk between IL31RA and other signaling pathways?

IL31RA signaling intersects with multiple pathways:

Experimental Strategies:

• Proximity ligation assays:

  • Detect protein-protein interactions between IL31RA and OSMR

  • Investigate interactions with JAK1/2, STAT3/5/1, PI3K components

  • Determine how these interactions change upon IL-31 stimulation

• Phosphoproteomic analysis:

  • Stimulate cells with IL-31

  • Perform phosphoproteomic analysis to identify activated pathways

  • Compare with other cytokine stimulations to identify unique signaling nodes

• Combined inhibition studies:

  • Use IL31RA blocking antibodies in combination with inhibitors of JAK/STAT, PI3K/AKT, or MAPK pathways

  • Assess synergistic or antagonistic effects on downstream biological responses

  • Monitor key phosphorylation events by western blotting

• Receptor complex analysis:

  • Use IL31RA antibodies for immunoprecipitation

  • Perform mass spectrometry to identify novel interacting partners

  • Validate these interactions using reverse co-immunoprecipitation

Research has shown that IL-31 signaling can activate JAK1, JAK2, STAT1, STAT3, STAT5, PI3K/AKT, and MAPK pathways, with the recruitment of SHP-2 and Shc adapter molecules contributing to increased MAPK pathway activation .

How can IL31RA antibodies be utilized to investigate the role of IL-31 signaling in cancer progression?

Recent research has implicated IL31RA in certain cancers, particularly basal-like breast cancer (BLBC):

Experimental Approaches:

• Expression analysis in cancer tissues:

  • Use IHC with IL31RA antibodies to assess expression in tumor samples

  • Compare expression between tumor and adjacent normal tissues

  • Correlate expression with clinical parameters and patient outcomes

• Functional studies in cancer cell lines:

  • Silence IL31RA using shRNA in cancer cell lines (e.g., MDA-MB-231 for BLBC)

  • Block IL31RA using neutralizing antibodies

  • Assess effects on:

    • Cancer stem cell-like properties (tumorsphere formation)

    • Cell migration (wound healing assay)

    • Invasion (transwell invasion assay)

    • Proliferation (growth curves, BrdU incorporation)

    • Signaling (phosphorylation of STAT3)

• In vivo tumor models:

  • Inject IL31RA-knockdown cancer cells into mice

  • Treat tumor-bearing mice with IL31RA blocking antibodies

  • Monitor tumor growth and metastasis

  • Analyze tumors for stemness and invasiveness markers

• Mechanistic studies:

  • Investigate transcriptional regulation of IL31RA in cancer (ChIP assays)

  • Identify downstream targets using RNA-seq after IL31RA modulation

  • Explore the role of STAT3 activation in mediating IL31RA effects

Research has shown that IL31RA promotes basal-like breast cancer progression and metastasis, suggesting that targeting the IL-31/IL31RA axis might be beneficial for BLBC treatment .

What methods can be used to investigate the differential roles of IL31RA isoforms in disease pathogenesis?

The multiple isoforms of IL31RA have distinct signaling capabilities and biological functions:

Research Strategies:

• Isoform-specific knockdown:

  • Design siRNAs targeting unique regions of each isoform

  • Verify knockdown specificity by qRT-PCR and western blot

  • Assess functional consequences on signaling and biological responses

• Isoform overexpression:

  • Clone the different isoforms (long 745 aa, short 560 aa, etc.)

  • Express in relevant cell types

  • Compare downstream signaling (particularly STAT activation)

  • Assess biological responses (cytokine production, proliferation, etc.)

• Isoform ratio analysis in disease:

  • Perform qRT-PCR with isoform-specific primers on samples from patients with various diseases (e.g., atopic dermatitis, psoriasis)

  • Compare the ratio of long (signaling) to short (inhibitory) forms

  • Correlate with disease severity and response to therapy

• Combined receptor analysis:

  • Assess co-expression of IL31RA isoforms with OSMR

  • Determine how the ratio of different forms and their co-expression with OSMR determines cellular responses to IL-31

Research has shown that a long (745 aa) and a short (560 aa) transmembrane form are the predominant forms, with many cell lines expressing both. The long form signals by recruiting STAT3, 5, or 1, while the short form does not recruit STATs and inhibits IL-31 signaling .

What are common challenges in detecting IL31RA and how can they be addressed?

Challenge 1: Low Basal Expression Levels

• Solution: Pre-treat cells with IFN-gamma (50 ng/mL for 20 hours) and bacterial lipopolysaccharides to upregulate IL31RA expression

• Alternative: Focus on cell types with known higher expression (dorsal root ganglia neurons, keratinocytes, macrophages)

Challenge 2: Multiple Isoforms Complicating Interpretation

• Solution: Use gradient gels for better separation of isoforms

• Alternative: Employ isoform-specific primers for PCR-based detection

• Verification: Use cell lines expressing known isoforms as controls

Challenge 3: Non-specific Antibody Binding

• Solution: Validate antibody specificity using IL31RA-knockdown cells

• Alternative: Compare multiple antibodies targeting different epitopes

• Control: Include appropriate isotype controls for flow cytometry and IHC

Challenge 4: Post-translational Modifications Affecting Detection

• Solution: Consider deglycosylation treatment before western blotting

• Alternative: Use antibodies targeting different domains/epitopes

• Analysis: Be aware that glycosylation may cause size variations in western blot

How should I interpret conflicting IL31RA expression data between different detection methods?

When facing discrepancies between detection methods:

• Consider method sensitivity differences:

  • qRT-PCR detects mRNA but not protein levels

  • Western blot may not detect low abundance proteins

  • Flow cytometry measures surface expression but may miss intracellular pools

  • IHC might show localization but has lower quantitative accuracy

• Evaluate technical factors:

  • Antibody epitope accessibility may differ between methods

  • Fixation can affect epitope recognition in IHC/ICC

  • Sample preparation may disrupt protein complexes or structure

• Biological considerations:

  • Different isoforms may be preferentially detected by certain methods

  • Post-translational modifications may affect antibody binding

  • Subcellular localization might influence detection (membrane vs. cytoplasmic)

  • Receptor internalization upon ligand binding may affect surface detection

• Resolution strategies:

  • Use multiple antibodies targeting different epitopes

  • Combine protein and mRNA detection methods

  • Include positive and negative controls for each method

  • Consider functional assays to validate expression findings

How can I differentiate between specific IL31RA antibody effects and off-target effects in functional studies?

When using IL31RA antibodies to block signaling:

• Essential controls:

  • Include isotype-matched control antibodies

  • Use IL31RA-knockdown or knockout cells alongside antibody treatment

  • Compare multiple antibodies targeting different IL31RA epitopes

  • Test dose-dependency of observed effects

• Validation approaches:

  • Confirm target engagement using binding assays

  • Verify pathway inhibition through phospho-STAT3 western blots

  • Use specific readouts of IL-31 signaling (e.g., STAT3 phosphorylation)

  • Compare with selective JAK inhibitors or other pathway blockers

• Rescue experiments:

  • Overexpress IL31RA to compete with antibody binding

  • Use mutated IL31RA constructs resistant to antibody recognition

  • Activate downstream pathways to bypass receptor inhibition

• Addressing cross-reactivity:

  • Test effects on cells lacking IL31RA expression

  • Assess potential binding to related receptors (e.g., other cytokine receptors)

  • Pre-absorb antibodies with recombinant IL31RA before functional studies

What are the key considerations when using IL31RA antibodies to study receptor-ligand interactions and signaling dynamics?

To accurately investigate IL-31/IL31RA interactions:

• Receptor complex formation:

  • Remember that functional IL-31 receptor is a heterodimer of IL31RA and OSMR

  • Verify expression of both components in your experimental system

  • Consider the ratio of different IL31RA isoforms and their co-expression with OSMR

• Binding kinetics:

  • Use surface plasmon resonance or similar techniques with purified components

  • Ensure antibodies don't interfere with natural binding kinetics if studying ligand interactions

  • Consider using non-blocking antibodies for detection purposes

• Signaling dynamics:

  • Monitor temporal activation of downstream pathways (JAK/STAT, PI3K/AKT, MAPK)

  • Assess how different IL31RA isoform ratios affect signaling outcomes

  • Investigate cross-talk with other cytokine receptors

• Cell-specific responses:

  • Compare signaling in different cell types (neurons vs. immune cells vs. epithelial cells)

  • Assess how microenvironment affects receptor expression and signaling

  • Investigate context-specific outcomes (e.g., pruritus in neurons, inflammatory responses in immune cells)

• Technical considerations:

  • Use antibodies recognizing the extracellular domain for blocking studies

  • Consider tagged recombinant ligands for binding studies

  • Employ real-time imaging techniques to visualize receptor dynamics

  • Use BiFC or FRET to study receptor dimerization and complex formation

What emerging research areas involving IL31RA antibodies should researchers be aware of?

Several promising research directions are developing:

• IL31RA in fibrotic diseases:

  • Recent research has implicated IL-31 signaling in fibrosis and Th2 polarization in systemic sclerosis

  • Blockade of IL-31 with anti-IL31RA antibody significantly ameliorated fibrosis in experimental models

  • IL31RA antibodies could be valuable tools to study mechanisms of fibrosis in various conditions

• Neuroimmunomodulatory functions:

  • IL31RA has been shown to play unexpected roles in restraining type 2 inflammation via neurogenic mechanisms

  • IL-31 activation of IL31RA+ neurons can trigger CGRP release, modulating immune responses

  • This represents a novel neuroimmune regulatory pathway worth exploring

• Cancer immunobiology:

  • IL31RA has been implicated in cancer stem cell-like properties and metastasis

  • The receptor may represent a therapeutic target in specific cancer types

  • Understanding how IL31RA regulates the tumor microenvironment is an emerging area

• Therapeutic antibody development:

  • IL31RA-targeting therapeutic antibodies like nemolizumab show promise in treating atopic dermatitis

  • Understanding the mechanisms behind clinical responses and potential resistance mechanisms

  • Investigating paradoxical responses seen in some patients

How might single-cell approaches enhance our understanding of IL31RA biology?

Single-cell technologies offer powerful ways to study IL31RA:

• Single-cell RNA sequencing:

  • Identify cell populations expressing IL31RA and its isoforms with high resolution

  • Discover rare IL31RA+ cell types that may be missed in bulk analyses

  • Characterize the co-expression patterns of IL31RA with other receptors and signaling components

  • Map the heterogeneity of IL31RA expression within seemingly homogeneous cell populations

• Single-cell proteomics:

  • Quantify IL31RA protein levels at single-cell resolution

  • Correlate IL31RA expression with other surface and intracellular proteins

  • Identify post-translational modifications affecting IL31RA function

• Spatial transcriptomics:

  • Map IL31RA expression in tissue contexts with spatial resolution

  • Understand the microenvironmental factors influencing IL31RA expression

  • Identify spatial relationships between IL31RA+ cells and other cell types

• Single-cell signaling analysis:

  • Use techniques like mass cytometry to measure IL-31-induced signaling at single-cell level

  • Identify differential responses to IL-31 within heterogeneous cell populations

  • Characterize how individual cells integrate IL-31 signals with other inputs

These approaches could reveal unprecedented insights into cell-specific roles of IL31RA in health and disease .

What interdisciplinary approaches might advance IL31RA research beyond current paradigms?

Combining expertise across fields could yield transformative insights:

• Neuroscience + Immunology:

  • Investigate how IL31RA+ neurons communicate with immune cells

  • Develop techniques to selectively activate or inhibit IL31RA+ neural circuits

  • Use optogenetics or chemogenetics to control IL31RA+ neurons and study downstream immune effects

• Structural Biology + Computational Modeling:

  • Determine the crystal structure of IL31RA/OSMR/IL-31 complex

  • Use this information to design better blocking antibodies or small molecules

  • Model receptor dynamics and predict effects of mutations or antibody binding

• Systems Biology + Machine Learning:

  • Integrate multi-omics data to build predictive models of IL31RA signaling networks

  • Identify key nodes and potential therapeutic targets

  • Predict patient responses to IL31RA-targeting therapies based on molecular profiles

• Tissue Engineering + Organoid Technology:

  • Develop complex 3D models incorporating IL31RA+ neurons, immune cells, and target tissues

  • Study IL-31 signaling in physiologically relevant microenvironments

  • Test IL31RA antibodies in these systems for better translation to in vivo contexts

• Clinical Biomarker Development + Personalized Medicine:

  • Identify biomarkers predicting response to IL31RA-targeting therapies

  • Develop companion diagnostics for treatment selection

  • Stratify patients based on IL31RA isoform expression profiles or pathway activation status