IL22RA1 Antibody

What is IL22RA1 and what is its primary biological function?

IL22RA1 is a type I transmembrane glycoprotein that belongs to the type II cytokine receptor family. It functions as a high-affinity ligand binding subunit for IL-22 and forms heterodimeric receptor complexes with either IL-10R beta or IL-20R beta, each with distinct ligand selectivities. The IL22RA1/IL-10R beta complex responds to IL-22, while the IL22RA1/IL-20R beta complex responds to IL-20 or IL-24 .

The primary biological function of IL22RA1 is to mediate signaling that promotes innate immune responses and wound healing at sites of infection and inflammation. This includes upregulation of antimicrobial peptides, acute phase proteins, proinflammatory cytokines, and extracellular matrix proteins, as well as regulation of proteases . Interestingly, IL22RA1 signaling also promotes downregulation of proteins involved in keratinocyte differentiation .

Which tissues and cell types express IL22RA1?

Unlike many cytokine receptors, IL22RA1 is not expressed on hematopoietic cells. Instead, its expression is restricted to epithelial and stromal cells . IL22RA1 is expressed in a limited number of tissues including:

• Skin epithelium

• Colon epithelial cells

• Liver (hepatocytes)

• Lung epithelial cells

• Pancreas

• Small intestine (including Paneth cells)

Recent single-cell studies have confirmed that approximately 72.7% of Paneth cells express Il22ra1 . The receptor is also found in various cell lines including HepG2 (human liver cancer cells), Hepa 1-6 (mouse hepatoma cells), and C2C12 cells (mouse myoblast cell line) .

What are the most common applications for IL22RA1 antibodies in research?

IL22RA1 antibodies are versatile research tools with multiple applications:

Different antibody clones may have varying specificities and applications, so it's essential to select an antibody validated for your specific experimental needs .

How should IL22RA1 antibodies be optimized for flow cytometry experiments?

For optimal flow cytometry results with IL22RA1 antibodies:

• Titration is crucial: Start with approximately 0.40 μg of antibody per 10^6 cells in a 100 μl suspension, but optimize this concentration for your specific cell type .

• Appropriate controls: Use isotype controls (such as MAB006 for rat antibodies or appropriate IgG controls for rabbit antibodies) to establish gating boundaries and determine non-specific binding .

• Secondary antibody selection: When using unconjugated primary antibodies, choose appropriate secondary antibodies such as Allophycocyanin-conjugated Anti-Rat IgG F(ab')2 or Phycoerythrin-conjugated Anti-Rat IgG, depending on your detection system .

• Sample preparation protocol: Follow established protocols for membrane-associated proteins, as IL22RA1 is a transmembrane receptor. Consider using protocols that preserve membrane integrity .

• Validation: Confirm specificity using known positive cell lines such as Hepa 1-6 mouse hepatoma cells or HepG2 human hepatocellular carcinoma cells, which have been demonstrated to express IL22RA1 .

What are the recommended approaches for using IL22RA1 antibodies in knockout/knockdown validation studies?

When using IL22RA1 antibodies to validate knockout or knockdown models:

• Multiple validation methods: Combine techniques such as PCR with recombination-specific primers and flow cytometry with IL22RA1-specific antibodies to confirm knockdown efficiency .

• Controls for conditional knockouts: For tissue-specific knockout models (e.g., Il22ra1^fl/fl;Villin-cre+ or Il22ra1^fl/fl;Defa6-cre+), use littermate Cre-negative floxed mice as wild-type controls .

• Functional validation: Beyond confirming reduced IL22RA1 expression, assess downstream effects such as changes in STAT3 phosphorylation or expression of IL22-responsive genes like antimicrobial peptides .

• Rescue experiments: Consider including IL-22 antibody treatment groups to verify that observed phenotypes are specifically due to IL-22/IL22RA1 signaling. This approach can help distinguish between direct and indirect effects of IL22RA1 deletion .

• Cell sorting strategies: For models with cell type-specific knockouts (e.g., Lgr5+ intestinal stem cells), use cell sorting based on reporter expression (such as GFP) to isolate the specific population for validation .

What controls are essential when using IL22RA1 antibodies for Western blotting?

For reliable Western blotting results with IL22RA1 antibodies:

• Positive controls: Include lysates from cells known to express IL22RA1, such as HepG2 cells or mouse liver tissue .

• Negative controls: Consider using lysates from hematopoietic cells, which do not express IL22RA1, as negative controls .

• Molecular weight verification: Confirm bands at the expected molecular weight of 63-68 kDa for the full-length protein, but be aware that post-translational modifications may affect migration .

• Dilution optimization: Start with dilutions between 1:1000-1:4000, but optimize based on your specific antibody and sample type .

• Loading controls: Use appropriate housekeeping proteins as loading controls to normalize expression levels.

• Specificity validation: Consider including lysates from siRNA-treated cells or tissue-specific knockout models as additional specificity controls .

How does the IL22RA1/JAK/STAT signaling pathway contribute to cancer progression?

IL22RA1 signaling has emerged as a significant factor in cancer development and progression:

What methodologies can be used to study IL22RA1's role in fibrosis and tissue remodeling?

To investigate IL22RA1's role in fibrosis and tissue remodeling:

• Human Tenon's capsule fibroblast (HTF) model: HTFs can be used to study fibroblast proliferation and activation in response to IL-22. Researchers can monitor cell cycle progression from G1 to S phase and measure α-SMA expression as indicators of myofibroblast differentiation .

• Gene knockdown/overexpression approaches: siRNA-mediated knockdown of IL22RA1 or overexpression systems can help determine whether IL-22's effects on fibrosis are directly mediated through IL22RA1 .

• Rescue experiments: Using IL-22 neutralizing antibodies in combination with IL22RA1 knockdown can help establish the specificity of observed effects .

• Cell cycle analysis: Flow cytometry can be used to quantify the proportion of cells in different cell cycle phases (G1, S, G2/M) following IL-22 treatment or IL22RA1 modulation .

• Protein expression analysis: Western blotting for IL22RA1 and fibrosis markers like α-SMA can track activation of fibrotic pathways .

What is the current understanding of IL22RA1's role in intestinal epithelial homeostasis?

IL22RA1 plays a crucial role in intestinal epithelial homeostasis:

• Paneth cell development: IL22RA1 signaling is critical for Paneth cell maturation. Studies using intestinal epithelium-specific knockout mice (Il22ra1^fl/fl;Villin-cre+) have shown defects in Paneth cell development and antimicrobial activity .

• Cell type-specific requirements: Interestingly, while IL22RA1 is required in Paneth cells, its expression in Lgr5+ intestinal stem cells appears to be dispensable for the development of secretory cells including Paneth cells .

• Antimicrobial defense: IL22RA1 signaling in intestinal epithelial cells induces expression of antimicrobial peptides and proteins that contribute to mucosal defense against pathogens .

• STAT3 dependency: The effects of IL-22 on intestinal epithelial cells are largely mediated through STAT3 activation downstream of IL22RA1 .

• Bacterial infection models: In models of intra-abdominal Klebsiella pneumoniae infection, recombinant IL-22 treatment shows therapeutic potential that depends on hepatic IL22RA1 and STAT3, resulting in potent bacteriostatic activity in serum .

Why might I detect different molecular weights for IL22RA1 in Western blotting experiments?

Variations in IL22RA1 molecular weight observed in Western blotting can be attributed to several factors:

• Expected molecular weight range: The calculated molecular weight of IL22RA1 is approximately 63 kDa, but it is typically observed between 63-68 kDa in Western blots .

• Post-translational modifications: IL22RA1 is a glycoprotein that undergoes N-linked glycosylation, which can increase its apparent molecular weight .

• Species differences: Mouse and human IL22RA1 may migrate slightly differently due to sequence variations and differences in post-translational modifications. Within the extracellular domain, mouse IL22RA1 shares 78% amino acid sequence identity with human IL22RA1 .

• Isoforms and splice variants: Alternative splicing may generate different isoforms with varying molecular weights.

• Sample preparation: The method of cell lysis and protein denaturation can affect the apparent molecular weight. Ensure complete denaturation of the protein by heating samples adequately in the presence of reducing agents.

• Gel percentage: The percentage of acrylamide in the gel can affect protein migration. Higher percentage gels provide better resolution for lower molecular weight proteins, while lower percentage gels are better for resolving higher molecular weight proteins.

How can I address cross-reactivity issues when using IL22RA1 antibodies?

To minimize cross-reactivity problems with IL22RA1 antibodies:

• Select antibodies with validated specificity: Choose antibodies that have been validated for specificity using knockout models or siRNA knockdown approaches .

• Consider epitope location: IL22RA1 shares 20-26% amino acid sequence identity with other class II cytokine receptors like IL-10R, IL-20R, and IL-28R . Select antibodies targeting unique epitopes to minimize cross-reactivity.

• Validation in multiple applications: An antibody that works well in one application may not be specific in others. Validate antibodies for each specific application .

• Appropriate controls: Include positive controls (known IL22RA1-expressing cells/tissues) and negative controls (hematopoietic cells that do not express IL22RA1) .

• Block non-specific binding: Use appropriate blocking agents to reduce background and non-specific binding.

• Antibody dilution optimization: Titrate antibodies to find the optimal concentration that maximizes specific signal while minimizing background and cross-reactivity .

What are the key considerations when designing experiments to study IL-22/IL22RA1 signaling in disease models?

When designing experiments to study IL-22/IL22RA1 signaling in disease contexts:

• Cell type specificity: Remember that IL22RA1 is not expressed on hematopoietic cells but is restricted to epithelial and stromal cells. Design experiments accordingly to focus on appropriate target cells .

• Receptor complex heterogeneity: IL22RA1 can form heterodimeric complexes with either IL-10RB or IL-20RB. Consider that different complexes may mediate different biological effects and may be differentially expressed across tissues .

• IL-22 binding protein (IL-22BP): Be aware that IL-22BP (also known as IL22RA2) functions as a competitive antagonist by binding IL-22 and preventing its association with IL22RA1. Consider measuring IL-22BP levels when studying IL-22/IL22RA1 signaling .

• Conditional knockout models: For in vivo studies, consider tissue-specific knockout models (e.g., Il22ra1^fl/fl;Villin-cre+ for intestinal epithelium) rather than global knockouts to avoid developmental effects and better model tissue-specific functions .

• Downstream pathway analysis: Include assessment of JAK/STAT pathway activation, particularly STAT3 phosphorylation, as well as MAPK and Akt pathway activation to fully characterize signaling events .

• Functional readouts: Incorporate appropriate functional assays relevant to the tissue being studied, such as antimicrobial peptide production in intestinal models or bacteriostatic activity in infection models .