P2RY6 Antibody

What is P2RY6 and what primary functions does it serve in biological systems?

P2RY6 (P2Y purinoceptor 6) is a G-protein coupled receptor that primarily binds UDP. It belongs to the P2Y receptor family within the larger G-protein coupled receptor superfamily. P2RY6 functions mainly through:

• Coupling to phospholipase C via Gq

• Inhibiting N-type Ca²⁺ and M-type K⁺ channels

• Inducing inositol phosphate/Ca²⁺ responses that are insensitive to pertussis toxin inhibition

• Mediating contractions in human cerebral arteries

• Amplifying pro-inflammatory responses, particularly in microglia

• Modulating metabolism and energy expenditure in adipose tissue

P2RY6 expression has been documented in various tissues including blood cells, bone marrow, brain, colon, heart, kidney, ovary, placenta, spleen, thymus, lung, stomach, intestine, and aorta .

What are the most reliable applications for P2RY6 antibodies in experimental research?

Based on multiple commercial antibody validations, P2RY6 antibodies are most reliably used in:

• Western blot analysis (typically at 1:200-1:500 dilution)

• Immunohistochemistry on paraffin or frozen sections (1:50-1:600 dilution)

• Immunofluorescence/immunocytochemistry (0.25-2 μg/mL)

For optimal results, researchers should note that:

• Pre-incubation with P2RY6 receptor blocking peptide can be used to confirm specificity

• Both N-terminal and C-terminal targeting antibodies are available, with different epitope recognition profiles

• Secondary detection systems commonly used include biotin-streptavidin methods with fluorescent conjugates .

What experimental controls are essential when using P2RY6 antibodies?

Critical controls for P2RY6 antibody experiments include:

• Peptide competition controls: Pre-incubating the antibody with the immunizing peptide should abolish specific signals.

• Knockout validation: Testing the antibody on tissue from P2RY6 knockout animals is the gold standard for specificity.

• Expression system controls: Testing on cells with confirmed expression versus cells lacking P2RY6.

• Multiple antibody approach: Using antibodies targeting different epitopes of P2RY6 to confirm findings.

• Orthogonal validation: Correlating protein detection with mRNA expression data.

Research has shown that relying solely on peptide competition is insufficient, as some commercial P2RY6 antibodies still produce signals in knockout tissues despite passing peptide competition tests .

How can researchers distinguish between non-specific binding and true P2RY6 signals in their antibody-based experiments?

To distinguish between non-specific binding and true P2RY6 signals:

• Implement rigorous validation protocols:

  • Knockout tissue validation is the gold standard

  • Heterologous expression systems can serve as positive controls

  • RNAi knockdown can provide partial validation

• Technical optimization strategies:

  • Titrate antibody concentration to minimize background

  • Modify blocking conditions (5% BSA often superior to serum for GPCRs)

  • Extend wash steps to reduce non-specific binding

  • Use detergent optimization for membrane proteins

• Analytical approaches:

  • Compare staining patterns across multiple tissues with known expression profiles

  • Correlate with functional data (e.g., UDP responses)

  • Use orthogonal detection methods (mRNA analysis)

Research has demonstrated that even "clean" Western blot bands can be misleading, as three commercially available antibodies to P2RY6 produced similar banding patterns in both wild-type and P2RY6 knockout tissue .

What are the signaling mechanisms through which P2RY6 exerts its functions in different cell types?

P2RY6 signaling mechanisms vary by cell type but generally involve these pathways:

In microglia, P2RY6-mediated signaling specifically enhances TLR-induced pro-inflammatory responses, with MRS2578 (a selective P2RY6 antagonist) demonstrating that P2RY6 acts primarily through modulation of NFAT, IRF and NF-κB transcription factors .

How does P2RY6 receptor activation interface with CCL2-CCR2 signaling in monocytes and what methodologies best capture this interaction?

P2RY6 receptor activation serves as a critical co-activator during CCL2-CCR2 signaling in monocytes:

• Mechanism of interaction:

  • P2RY6 activation accounts for approximately 80% of the intracellular Ca²⁺ signal evoked by CCL2

  • THP-1 cells rapidly secrete ATP during signaling downstream of the CCL2-CCR2 axis

  • This ATP release acts as a mechanism for P2Y6 receptor co-activation

• Experimental approaches to study this interaction:

  • Nucleotide scavenging with apyrase (causes fourfold reduction in THP-1 sensitivity to CCL2)

  • Inhibition of CD39-like ectonucleotidases (potentiates CCL2-evoked Ca²⁺ responses)

  • Pharmacological inhibition of P2Y6 (impairs CCL2-evoked Ca²⁺ signaling and chemotaxis)

  • P2Y6 receptor knockdown (impairs CCL2-evoked Ca²⁺ signaling, chemotaxis and adhesion to TNFα-treated HUVECs)

• Quantification methods:

  • Calcium imaging to measure P2RY6 contribution to CCL2 responses

  • Chemotaxis assays with stable knockdown systems

  • Adhesion assays with endothelial cell models

This research demonstrates that P2RY6 receptor mediates leukocyte responsiveness to CCL2, representing a novel mechanism by which to modulate CCL2 signals .

What technical considerations are critical when validating P2RY6 antibodies across different species?

When validating P2RY6 antibodies across species, researchers should address these technical considerations:

• Epitope conservation analysis:

  • Compare sequence homology at the antibody binding region

  • For example, antibody #APR-011 targets amino acid residues 311-328 of rat P2Y6 receptor (CQPHDLLQKLTAKWQRQRV)

  • NBP2-82301 targets the N-terminal region (NLHGSILFLTCISFQRYLGICHPLAPWHKRGGRRAAWLVCVAVWLAVTTQ)

  • Cross-species reactivity predictions should be verified experimentally

• Species-specific validation approaches:

  • Use tissues with confirmed expression profiles for each species

  • Implement knockout controls when available for each species

  • Consider species-specific post-translational modifications

• Application-specific optimization:

  • Different dilutions may be required for the same antibody across species

  • Fixation protocols may need species-specific modification

  • Blocking conditions should be optimized for each species

• Prediction tools and experimental validation:

  • Some antibodies provide prediction scores for cross-reactivity

  • High confidence scores (>80) may suggest cross-reactivity, but experimental verification is essential

Multiple commercial antibodies report reactivity with human, mouse, and rat P2RY6, but researchers should independently verify this reactivity for their specific application .

How can researchers effectively study the role of P2RY6 in metabolic regulation and what phenotypic changes should be monitored?

To effectively study P2RY6's role in metabolic regulation:

• Experimental models:

  • Adipocyte-specific P2RY6 knockout mice

  • Whole-body P2RY6 knockout mice

  • P2RY6 antagonist (MRS2578) treatment studies

  • Cell-specific knockdown of P2RY6 in metabolic tissues

• Key phenotypic parameters to monitor:

  • Body weight and composition (fat/lean mass ratio)

  • Energy expenditure (indirect calorimetry)

  • Glucose tolerance and insulin sensitivity

  • Adipose tissue inflammation markers

  • White adipose tissue browning (UCP1, PGC1α expression)

  • JNK signaling pathway components

  • PPARα activity assessment

• Molecular mechanisms to investigate:

  • JNK signaling and activity measurements

  • PPARα expression and downstream targets

  • PGC1α levels in adipose tissue

  • Inflammatory cytokine profiles

Research has demonstrated that adipocyte-specific deletion of P2Y6R protects mice from diet-induced obesity through enhanced energy expenditure, reduced inflammation, and white adipose tissue browning, associated with reduced JNK signaling and enhanced PPARα activity .

What approaches can be taken to address contradictory findings regarding P2RY6 antibody specificity in the literature?

To address contradictory findings regarding P2RY6 antibody specificity:

• Implement comprehensive validation strategies:

  • Use genetic knockout validation as the gold standard

  • Compare multiple antibodies targeting different epitopes

  • Employ orthogonal detection methods (mRNA, functional assays)

  • Conduct heterologous expression studies in null backgrounds

• Critically evaluate published literature:

  • Assess validation methods used in published studies

  • Note that peptide competition alone is insufficient validation

  • Consider that even "clean" Western blot bands may be misleading

  • Evaluate whether knockout controls were implemented

• Develop standardized reporting:

  • Document all validation steps comprehensively

  • Report negative findings regarding antibody specificity

  • Provide detailed methodology for reproducibility

Evidence from knockout validation studies shows that some widely used commercial P2RY6 antibodies (including APR-011 from Alomone Labs) produce identical staining patterns in both wild-type and P2RY6 knockout tissues, despite producing clean Western blot bands that are eliminated by peptide competition .

How can P2RY6 signaling be targeted therapeutically in inflammatory and metabolic diseases?

Therapeutic targeting of P2RY6 signaling shows promise for inflammatory and metabolic diseases:

• Pharmacological approaches:

  • Selective P2RY6 antagonists (e.g., MRS2578)

  • Development of novel antagonists with improved pharmacokinetics

  • Cell-type selective delivery approaches

• Target diseases and conditions:

  • Obesity and type 2 diabetes (adipocyte-specific targeting)

  • Neuroinflammatory conditions (microglial targeting)

  • Inflammatory vascular diseases

• Experimental readouts for therapeutic efficacy:

  • Pro-inflammatory cytokine reduction in microglia

  • Metabolic improvements in adipose tissue

  • Energy expenditure enhancement

  • Insulin sensitivity improvement

• Combination strategies:

  • P2RY6 antagonism with TLR pathway modulators for neuroinflammation

  • P2RY6 inhibition with CCR2 antagonists for monocyte-driven inflammation

  • Metabolic therapies combining P2RY6 targeting with established anti-diabetic agents

Research on adipocyte-specific P2RY6 deletion suggests that P2RY6 antagonists may be beneficial for treating obesity and type 2 diabetes through multiple mechanisms including reduced inflammation, enhanced energy expenditure, and promotion of white adipose tissue browning .