Phospho-IL10RA (Tyr496) Antibody

What is IL-10RA and why is phosphorylation at Tyr496 significant?

The IL-10 receptor alpha (IL-10RA) is a subunit of the IL-10 receptor complex, a member of the class II subgroup of the cytokine receptor family that exhibits structural similarity to the interferon receptor. IL-10RA is primarily expressed in hematopoietic cells including B-cells, T-cells, NK cells, monocytes, and macrophages . It is not expressed in non-hematopoietic cells such as fibroblasts or endothelial cells .

Phosphorylation at Tyr496 is one of two critical tyrosine phosphorylation sites (along with Tyr446) in the cytoplasmic domain of IL-10RA. When IL-10 binds to its receptor, it activates JAK1 and Tyk2 tyrosine kinases, which subsequently phosphorylate these tyrosine residues . The phosphorylation of Tyr496 is essential for proper IL-10R function and activation of downstream IL-10 signaling pathways, playing a crucial role in regulating immune responses and inflammation .

How does IL-10RA phosphorylation contribute to immune regulation?

IL-10 is a potent anti-inflammatory cytokine that inhibits the production of pro-inflammatory cytokines. When IL-10 binds to its receptor, the phosphorylation of IL-10RA at Tyr496 initiates a signaling cascade that mediates the immunosuppressive effects of IL-10 . This signaling is critical for controlling excessive immune responses and maintaining immune homeostasis.

The phosphorylation of IL-10RA at Tyr496 is required for proper functioning of the IL-10 receptor and activation of downstream pathways, including STAT3 signaling. Defects in this phosphorylation can lead to dysregulated immune responses, potentially contributing to inflammatory conditions such as inflammatory bowel disease (IBD) . Studies have shown that mutations affecting IL-10 or its receptor can result in early-onset IBD, highlighting the importance of this signaling pathway in immune regulation .

What are the primary applications for Phospho-IL-10RA (Tyr496) antibodies in research?

Phospho-IL-10RA (Tyr496) antibodies are versatile tools with several key applications:

• Western Blot (WB): Used to detect and quantify phosphorylated IL-10RA at Tyr496 in cell or tissue lysates, allowing researchers to assess activation states of IL-10 signaling under various conditions .

• Immunofluorescence/Immunocytochemistry (IF/ICC): Enables visualization of the cellular localization and expression patterns of phosphorylated IL-10RA in intact cells .

• ELISA: Quantitative measurement of phosphorylated IL-10RA levels in complex biological samples, particularly useful for high-throughput screening .

• Cell-based assays: For monitoring the effects of various treatments, inhibitors, or activators on IL-10RA phosphorylation in living cells .

These applications allow researchers to investigate IL-10 signaling dynamics in various experimental contexts, from basic mechanistic studies to disease-relevant models.

What is the recommended protocol for detecting phospho-IL-10RA (Tyr496) by Western blot?

For optimal Western blot detection of phospho-IL-10RA (Tyr496):

• Sample preparation:

  • Stimulate cells with IL-10 to induce receptor phosphorylation

  • Lyse cells in buffer containing phosphatase inhibitors to preserve phosphorylation state

  • Denature proteins by heating in sample buffer containing SDS and reducing agent

• Gel electrophoresis and transfer:

  • Separate proteins on SDS-PAGE (typically 8-10% gel for IL-10RA's ~63kDa size)

  • Transfer to PVDF or nitrocellulose membrane

• Antibody incubation:

  • Block membrane with appropriate blocking buffer

  • Incubate with primary phospho-IL-10RA (Tyr496) antibody at dilution 1:500-1:1000

  • Wash thoroughly and incubate with appropriate HRP-conjugated secondary antibody

• Detection:

  • Develop using enhanced chemiluminescence (ECL) substrate

  • Expected band size is approximately 63 kDa

For accurate interpretation, include positive controls (IL-10-stimulated cells) and negative controls (unstimulated cells or phosphatase-treated samples).

What factors should be considered when selecting a phospho-IL-10RA (Tyr496) antibody?

When selecting a phospho-IL-10RA (Tyr496) antibody, researchers should consider:

• Specificity: Ensure the antibody specifically recognizes IL-10RA phosphorylated at Tyr496 without cross-reactivity to unphosphorylated IL-10RA or other phosphorylated proteins. Antibodies raised against synthetic phosphopeptides derived from the region surrounding Tyr496 often provide high specificity .

• Host species: Consider the host species (typically rabbit for available products) to avoid cross-reactivity in your experimental system .

• Applications: Verify the antibody has been validated for your specific application (WB, IF/ICC, ELISA) . Some antibodies may perform better in certain applications than others.

• Species reactivity: Check if the antibody reacts with your species of interest. Many phospho-IL-10RA antibodies are reactive with human samples, with predicted reactivity to other species like pig, horse, rabbit, dog, and chicken .

• Clonality: Most phospho-IL-10RA (Tyr496) antibodies are polyclonal, which can provide higher sensitivity but potentially lower specificity compared to monoclonal antibodies .

• Validation data: Review available validation data, including Western blot images and control experiments, to ensure reliable performance.

How should phospho-IL-10RA (Tyr496) antibodies be stored and handled?

Proper storage and handling are crucial for maintaining antibody performance:

• Storage conditions:

  • Short-term (up to 1 week): Store at 4°C

  • Long-term: Store at -20°C, typically in small aliquots to avoid repeated freeze-thaw cycles

  • Many commercial antibodies are supplied in a stabilizing buffer containing preservatives such as sodium azide and glycerol

• Working dilutions:

  • For Western blot: Typical dilutions range from 1:500 to 1:1000

  • Optimal dilutions should be determined empirically for each application and experimental setup

• Stability considerations:

  • Avoid repeated freeze-thaw cycles which can degrade antibody performance

  • Prepare working dilutions fresh before use

  • Follow manufacturer's recommendations for specific products

• Safety precautions:

  • Many antibody solutions contain sodium azide, which is toxic and should be handled accordingly

What are common issues when detecting phospho-IL-10RA (Tyr496) and how can they be addressed?

Researchers often encounter several challenges when working with phospho-specific antibodies:

• Low signal intensity:

  • Ensure proper IL-10 stimulation to induce phosphorylation

  • Include phosphatase inhibitors in all buffers

  • Optimize antibody concentration and incubation time

  • Consider using signal enhancement systems

• High background:

  • Increase blocking time or concentration

  • Use more stringent washing conditions

  • Reduce primary and secondary antibody concentrations

  • Ensure blocking buffer is compatible with phospho-epitopes

• Multiple bands:

  • Verify sample preparation to minimize protein degradation

  • Use freshly prepared lysates

  • Optimize SDS-PAGE conditions

  • Consider that IL-10RA may exist in different glycosylation states

• Loss of phospho-signal:

  • Minimize time between sample collection and analysis

  • Keep samples cold during processing

  • Use fresh phosphatase inhibitors in all buffers

  • Avoid repeated freeze-thaw cycles of samples

• Inconsistent results:

  • Standardize stimulation protocols

  • Use consistent cell densities and passage numbers

  • Include appropriate positive and negative controls

  • Normalize phospho-IL-10RA signal to total IL-10RA

How can I validate the specificity of phospho-IL-10RA (Tyr496) antibody in my experimental system?

Rigorous validation is essential for phospho-specific antibodies:

• Phosphatase treatment:

  • Treat duplicate samples with lambda phosphatase to remove phosphate groups

  • Signal should disappear in phosphatase-treated samples

• Stimulation experiments:

  • Compare unstimulated cells with IL-10-stimulated cells

  • Signal should increase following IL-10 stimulation

• Kinase inhibitors:

  • Treat cells with JAK1/Tyk2 inhibitors before IL-10 stimulation

  • Signal should be reduced with inhibitor treatment

• Peptide competition:

  • Pre-incubate antibody with phosphorylated and non-phosphorylated peptides

  • Only the phosphorylated peptide should block antibody binding

• siRNA knockdown:

  • Reduce IL-10RA expression using siRNA

  • Both total and phospho-IL-10RA signals should decrease

• Mutant expression:

  • Express wild-type IL-10RA and Y496F mutant in a model system

  • Phospho-antibody should only detect wild-type protein after IL-10 stimulation

How can phospho-IL-10RA (Tyr496) antibodies be used to study IL-10 signaling dynamics?

Advanced applications for studying signaling dynamics include:

• Time-course experiments:

  • Monitor phosphorylation kinetics following IL-10 stimulation

  • Track receptor activation, peak phosphorylation, and dephosphorylation

  • Correlate with downstream signaling events (STAT3 phosphorylation, target gene expression)

• Dose-response studies:

  • Examine relationship between IL-10 concentration and receptor phosphorylation

  • Establish EC50 values for different cell types or under different conditions

  • Compare with physiological IL-10 concentrations in various disease states

• Single-cell analysis:

  • Use phospho-flow cytometry to assess heterogeneity in IL-10 responses

  • Combine with other markers to identify responsive cell populations

  • Correlate with functional outputs at the single-cell level

• Signaling crosstalk:

  • Investigate how other cytokine pathways influence IL-10RA phosphorylation

  • Study effects of inflammatory mediators on IL-10 responsiveness

  • Examine receptor phosphorylation in disease models

• Live-cell imaging:

  • Combine with proximity ligation assays to visualize phosphorylation events in situ

  • Track receptor clustering and internalization following phosphorylation

  • Monitor co-localization with signaling components

What role does IL-10RA Tyr496 phosphorylation play in disease pathogenesis?

The phosphorylation of IL-10RA at Tyr496 is critical for proper immune regulation, and dysregulation of this process may contribute to various diseases:

• Inflammatory bowel disease (IBD):

  • IL-10 and IL-10R deficiencies are associated with early-onset IBD

  • Defective IL-10RA phosphorylation may impair anti-inflammatory signals

  • Studying phosphorylation patterns in patient samples can provide insights into disease mechanisms

• Autoimmune disorders:

  • Aberrant IL-10 signaling contributes to dysregulated immune responses

  • Phospho-IL-10RA antibodies can help characterize receptor function in patient cells

  • Potential therapeutic target for enhancing anti-inflammatory signals

• Cancer immunology:

  • IL-10 has complex roles in tumor immunity

  • Monitoring IL-10RA phosphorylation in tumor-infiltrating immune cells

  • Investigating how cancer cells may exploit or evade IL-10 signaling

• Infectious diseases:

  • Some pathogens manipulate IL-10 signaling to evade immunity

  • Phospho-IL-10RA antibodies can reveal alterations in receptor activation during infection

  • Studying how microbial factors impact IL-10RA phosphorylation

Beyond antibody-based detection, what other methods can assess IL-10RA phosphorylation status?

While antibody-based methods are most common, alternative approaches include:

• Mass spectrometry:

  • Provides precise identification and quantification of phosphorylation sites

  • Can detect novel or unexpected phosphorylation events

  • Requires specialized equipment and expertise

• Proximity ligation assays:

  • Enables in situ detection of phosphorylated receptors

  • Provides spatial information about phosphorylation events

  • Higher sensitivity than conventional immunofluorescence

• CRISPR-based reporters:

  • Engineered cells expressing fluorescent proteins linked to phosphorylation-dependent domains

  • Allows real-time monitoring of signaling events

  • Requires genetic manipulation of target cells

• Bioluminescence resonance energy transfer (BRET):

  • Measures protein interactions dependent on phosphorylation

  • Enables real-time monitoring in live cells

  • Requires expression of fusion proteins

• Phospho-proteomic approaches:

  • Global analysis of phosphorylation changes in response to IL-10

  • Places IL-10RA phosphorylation in broader signaling context

  • Identifies novel components of IL-10 signaling networks

How does phospho-specific IL-10RA (Tyr496) antibody compare with total IL-10RA antibody in research applications?

The complementary use of phospho-specific and total IL-10RA antibodies provides comprehensive insights:

• Experimental design considerations:

  • Phospho-antibodies detect active signaling state

  • Total antibodies measure expression levels regardless of activation

  • Using both allows normalization of phosphorylation to expression levels

• Interpretation differences:

  • Changes in phospho-IL-10RA signal can indicate altered signaling efficiency

  • Changes in total IL-10RA reflect expression regulation

  • Ratio of phospho to total provides activation state independent of expression

• Technical distinctions:

  • Phospho-antibodies often require more careful sample handling

  • Total antibodies typically generate stronger signals

  • Different optimization parameters may be needed for each

• Combined applications:

  • Sequential probing of the same membrane

  • Parallel samples run with different antibodies

  • Dual immunofluorescence to simultaneously detect both forms