Phospho-CXCR2 (Ser347) Antibody

What is CXCR2 and what role does phosphorylation at Ser347 play?

CXCR2 is a receptor for interleukin-8, functioning as a powerful neutrophil chemotactic factor. It belongs to the G-protein coupled receptor (GPCR) family. Phosphorylation at Serine 347 is a post-translational modification that occurs following receptor activation. This phosphorylation is a key regulatory mechanism that controls CXCR2 desensitization, β-arrestin recruitment, and receptor internalization. The phosphorylation state at this residue is critical for regulating neutrophil chemotaxis and inflammatory responses.

How does the Phospho-CXCR2 (Ser347) Antibody differ from general CXCR2 antibodies?

Phospho-CXCR2 (Ser347) antibodies are specifically designed to detect CXCR2 only when phosphorylated at Serine 347, unlike general CXCR2 antibodies that recognize the receptor regardless of its phosphorylation state. This specificity allows researchers to monitor the activation status of the receptor, as phosphorylation at Ser347 occurs following ligand binding and receptor activation. The antibodies are typically validated using blocking peptides to confirm their specificity for the phosphorylated form.

What is the relationship between CXCR2 phosphorylation and neutrophil function?

When IL-8 binds to CXCR2, it triggers receptor activation, leading to phosphorylation at sites including Ser347. This phosphorylation mediates signal transduction via G-proteins that activate a phosphatidylinositol-calcium second messenger system. The resulting signaling cascade is crucial for neutrophil chemotaxis, respiratory burst, and degranulation. Phosphorylation at Ser347 specifically regulates desensitization mechanisms that prevent excessive or prolonged neutrophil responses, which is essential for controlled inflammatory reactions.

What are the validated applications for Phospho-CXCR2 (Ser347) antibodies?

Based on supplier information, Phospho-CXCR2 (Ser347) antibodies have been validated for multiple applications:

Researchers should optimize dilutions based on their specific experimental conditions and sample types.

How can I validate the specificity of phospho-specific CXCR2 antibodies in my experiments?

To validate phospho-specificity:

• Perform comparative analysis with and without phosphatase treatment of your samples

• Use blocking peptides (both phosphorylated and non-phosphorylated) to confirm specific recognition

• Stimulate cells with known CXCR2 agonists (e.g., IL-8, CXCL6) versus unstimulated controls to demonstrate inducible phosphorylation

• Include negative controls with PKC activators that don't induce phosphorylation at Ser347

• Employ paired antibodies (phospho-specific and total CXCR2) on the same samples to normalize for total receptor expression

Many suppliers provide blocking peptides specifically for validation purposes, as shown in several immunoblot examples where phospho-peptide competition abolishes antibody recognition.

What are the recommended sample preparation methods to preserve CXCR2 phosphorylation?

Preserving phosphorylation status is critical for accurate detection:

• Rapidly harvest and process samples to minimize dephosphorylation by endogenous phosphatases

• Include phosphatase inhibitor cocktails in all lysis and extraction buffers

• For cell stimulation experiments, use short time points (30 minutes shown to be effective for CXCL6 stimulation)

• Use cold PBS with phosphatase inhibitors for washing steps

• For tissue samples, use rapid fixation protocols with phosphatase inhibitors

• Avoid freeze-thaw cycles of protein lysates

• Maintain samples at 4°C throughout processing when possible

These steps are essential as phosphorylation is a labile modification that can be rapidly lost during sample handling.

How can phospho-CXCR2 (Ser347) antibodies be used to study biased signaling of CXCR2?

For studying biased signaling:

• Compare phosphorylation patterns induced by different CXCR2 ligands (IL-8, CXCL1, CXCL3, GRO/MGSA, NAP-2)

• Correlate Ser347 phosphorylation with β-arrestin recruitment using BRET or FRET assays

• Analyze G-protein dependent versus β-arrestin dependent signaling pathways

• Use time-course experiments to determine the kinetics of phosphorylation following stimulation with different ligands

• Combine with inhibitors of different GRK family members to identify kinase-specific phosphorylation mechanisms

The pT347-CXCR2 antibody has been demonstrated to detect phosphorylation in response to both high- and low-efficacy agonists but not after PKC activation, making it particularly useful for biased signaling studies.

What can phospho-CXCR2 antibodies reveal about receptor internalization and trafficking mechanisms?

To study receptor trafficking:

• Combine immunofluorescence using phospho-CXCR2 (Ser347) antibodies with markers of different endocytic compartments

• Perform time-course analysis of receptor phosphorylation, internalization, and recycling

• Co-stain with β-arrestin to analyze recruitment kinetics and colocalization following ligand stimulation

• Use phospho-mutant CXCR2 (S347A) transfected cells as controls

• Compare trafficking patterns of phosphorylated receptors versus total receptor population

Research has established that T347/S347 phosphorylation is a key regulator of CXCR2 desensitization, β-arrestin recruitment, and internalization, making these antibodies valuable tools for trafficking studies.

How do I troubleshoot inconsistent phospho-CXCR2 (Ser347) detection in neutrophil samples?

When troubleshooting neutrophil experiments:

• Ensure rapid isolation of neutrophils to prevent spontaneous activation and receptor phosphorylation

• Consider the half-life of neutrophils (short-lived cells) when designing experiments

• Monitor neutrophil activation status using flow cytometry for activation markers

• Test multiple cell lysis buffers optimized for membrane proteins

• For human neutrophil samples, minimize handling time and maintain samples at 4°C

• Verify antibody reactivity with your species (human and mouse reactivity are most commonly validated)

• Use positive controls (e.g., HEK293 cells expressing CXCR2 stimulated with CXCL6)

• Check for interference from other inflammatory mediators that might affect phosphorylation states

Figure 1 from source demonstrates successful detection in cell lines, providing a reference for expected results.

What are the differences between phospho-Ser347 and phospho-Thr347 CXCR2 antibodies?

The differences between these antibodies are important for experimental design:

Note that some literature refers to the same position as either S347 or T347, which may represent species-specific differences or nomenclature variations between suppliers.

How does species cross-reactivity affect experimental design with phospho-CXCR2 antibodies?

Species cross-reactivity considerations:

• Human CXCR2 antibodies may not recognize mouse or rat CXCR2 due to sequence variations around the phosphorylation site

• Verify reactivity claims with your experimental species (human and mouse are most commonly validated)

• Sequence alignment analysis shows good homology for human and monkey CXCR2, with variations in rodent sequences

• For pig, bovine, and dog samples, prediction scores from alignment analysis suggest potential reactivity, but experimental validation is necessary

• Use well-characterized positive controls from your species of interest

• Consider testing multiple antibodies from different suppliers if working with non-human/mouse species

The full CXCR2 protein sequence from UniProt ID P25025 can be useful for analyzing conservation of the phosphorylation site across species.

Can these antibodies distinguish between CXCR1 and CXCR2 phosphorylation?

This is crucial for studies of chemokine receptor specificity:

• Sequence alignment shows differences between CXCR1 and CXCR2 in the C-terminal region containing Ser347

• Phospho-CXCR2 (Ser347) antibodies are designed against synthetic phosphopeptides specific to CXCR2

• Cross-reactivity testing with phosphorylated CXCR1 is recommended before using in systems expressing both receptors

• Western blot analysis can help distinguish between the two receptors based on slight molecular weight differences

• In neutrophils expressing both receptors, use receptor-specific antagonists to confirm specificity of detected phosphorylation signals

No significant cross-reactivity with CXCR1 has been reported for the phospho-CXCR2 (Ser347) antibodies based on available information, but comprehensive validation is recommended for specific experimental contexts.

How can phospho-CXCR2 antibodies be integrated with phosphoproteomic workflows?

For integration with phosphoproteomics:

• Use phospho-CXCR2 (Ser347) antibodies for immunoprecipitation followed by mass spectrometry

• Compare targeted (antibody-based) versus global phosphoproteomic data to validate site-specific phosphorylation

• Perform temporal analysis of the CXCR2 phosphorylation barcode using multiple phospho-specific antibodies

• Employ phospho-CXCR2 (Ser347) detection to normalize or validate phosphoproteomic datasets

• Combine with inhibitor studies to establish kinase-substrate relationships for CXCR2 phosphorylation

This approach allows for comprehensive mapping of phosphorylation events in the CXCR2 signaling pathway.

What control samples should be included when studying CXCR2 phosphorylation in pathological contexts?

For pathological studies, include:

• Matched healthy tissue/cells from the same patient when possible

• Phosphatase-treated samples as negative controls

• Samples stimulated with known CXCR2 agonists as positive controls

• Isotype control antibodies to verify specificity of staining

• Competing phosphopeptide controls to confirm phospho-specificity

• For cancer studies, compare phosphorylation across different tumor grades and stages

• Include normal adjacent tissue in immunohistochemical analyses

Examples of successful controls include the use of competing phosphopeptides in breast carcinoma tissue analysis.

How can phospho-CXCR2 (Ser347) antibodies be used to evaluate therapeutic efficacy of CXCR2 antagonists?

For drug development applications:

• Establish baseline phosphorylation levels in disease models

• Monitor Ser347 phosphorylation as a biomarker of receptor activation status

• Combine with functional assays (chemotaxis, calcium flux) to correlate phosphorylation with receptor function

• Use dose-response and time-course analyses to determine antagonist efficacy

• Compare novel antagonists with reference compounds for relative efficacy

• Evaluate effects on receptor phosphorylation versus total receptor expression

• Analyze the relationship between receptor occupancy, phosphorylation inhibition, and functional outcomes

This approach provides molecular insights into mechanisms of action beyond simple binding assays.

What are the optimal storage conditions for phospho-CXCR2 antibodies to maintain activity?

For optimal storage:

Following these guidelines helps maintain antibody performance over time.

How should I optimize blocking conditions for phospho-specific Western blots?

For optimal Western blot results:

• Test different blocking agents (BSA is often preferred over milk for phospho-specific antibodies)

• Use 3-5% BSA in TBS-T for blocking (milk contains phosphatases that may reduce signal)

• Include phosphatase inhibitors in all buffers

• Optimize primary antibody incubation time (overnight at 4°C often yields best results)

• Use fresh transfer buffers with methanol to improve transfer of hydrophobic membrane proteins

• For PVDF membranes, a longer activation in methanol may improve results

• Consider using signal enhancers specifically designed for phospho-protein detection

Following manufacturer's recommended dilutions (typically 1:1000 for WB) provides a starting point, but optimization may be necessary for specific sample types.

What fixation methods are compatible with phospho-CXCR2 immunohistochemistry?

For immunohistochemical applications:

• Paraformaldehyde (4%) fixation preserves phospho-epitopes well

• Formalin-fixed paraffin-embedded (FFPE) tissues are compatible based on validated IHC-P applications

• Consider epitope retrieval methods (heat-induced epitope retrieval in citrate buffer pH 6.0)

• For frozen sections, brief fixation (10 min) in 4% paraformaldehyde is recommended

• Include phosphatase inhibitors in fixation buffers when possible

• Cold methanol fixation may not be optimal for preserving phospho-epitopes

• Test multiple fixation times if signal is weak or inconsistent

Examples of successful IHC applications on human breast carcinoma tissue have been documented using these approaches.