NPY6R Antibody

What is NPY6R and what are its known biological functions?

NPY6R (Neuropeptide Y receptor Y6) is a G-protein-coupled receptor belonging to the neuropeptide Y receptor family. Interestingly, when expressed, NPY6R is unable to bind pancreatic polypeptide (PP), neuropeptide Y (NPY), or peptide YY (PYY), suggesting that it may be functionally inactive or has potentially acquired a pancreatic polypeptide-independent function . NPY6R is also known by several alternative names including NPY1RL, Y2B, Putative neuropeptide Y receptor type 6, NPY6-R, NPY Y1-like receptor, and Putative pancreatic polypeptide receptor 2 (PP2) . While NPY receptors typically mediate effects of neuropeptide Y family peptides, the specific role of NPY6R requires further investigation as it appears to function differently from other NPY receptors .

What experimental techniques are validated for NPY6R antibodies?

Current research validates the use of NPY6R antibodies (such as ab223515) for several laboratory techniques:

• Immunocytochemistry/Immunofluorescence (ICC/IF): Validated at 1/100 dilution for cell lines such as MCF7

• Immunohistochemistry with paraffin-embedded sections (IHC-P): Effective at 1/100 dilution for human tissue samples

• Western blotting (WB): Suitable for protein analysis

When selecting a technique, researchers should consider the specific research question and available sample types, as different applications may require optimization of antibody concentration and detection methods.

What sample types have been validated with NPY6R antibodies?

Research demonstrates that polyclonal NPY6R antibodies (such as ab223515) have been validated for use with:

• Human samples: Particularly effective in kidney and skeletal muscle tissues, as well as MCF7 breast adenocarcinoma cells

• Mouse samples: Covered by product promise based on reactivity testing

• Rat samples: Covered by product promise based on reactivity testing

For tissues or species not explicitly validated, researchers should perform preliminary experiments with appropriate positive and negative controls to verify antibody specificity before proceeding with full experimental protocols.

How should researchers optimize dilution ratios for different NPY6R antibody applications?

Optimization of NPY6R antibody dilutions depends on the specific application:

• For ICC/IF applications: A 1/100 dilution has been validated for MCF7 cells with successful results using secondary antibodies such as Alexa Fluor 488® conjugated Goat Anti-Rabbit IgG

• For IHC-P applications: A 1/100 dilution has been successfully used in human kidney and skeletal muscle tissues

Researchers should consider performing a dilution series (e.g., 1/50, 1/100, 1/200, 1/500) during initial optimization to determine the optimal signal-to-background ratio for their specific samples and experimental conditions. Recording specific lot numbers and standardizing protocols is recommended for experimental reproducibility.

What expression pattern should researchers expect when using NPY6R antibodies?

Based on current research findings, NPY6R expression varies significantly across tissues and disease states:

• In normal tissues: Expression patterns should be validated by researchers as comprehensive normal tissue data is limited

• In cancer tissues: NPY6R is poorly expressed in most tumor types examined, with significant downregulation observed in multiple cancers including BLCA, BRCA, CESC, CHOL, COAD, ESCA, GBM, HNSC, LGG, LIHC, LUAD, LUSC, OV, PCPG, PRAD, READ, TGCT, THCA, UCEC, and UCS

• In uveal melanoma (UVM): NPY6R shows low expression levels compared to some normal tissues

When performing immunostaining, researchers should expect cellular localization patterns consistent with G-protein-coupled receptor expression, primarily at cellular membranes.

What controls should be included when working with NPY6R antibodies?

When designing experiments with NPY6R antibodies, researchers should incorporate the following controls:

• Positive controls: Use tissues or cell lines with known NPY6R expression such as MCF7 cells, human kidney tissue, or human skeletal muscle tissue

• Negative controls: Include samples without primary antibody incubation to assess background staining

• Isotype controls: Use matched isotype immunoglobulins at equivalent concentrations to rule out non-specific binding

• Blocking peptide controls: Where available, pre-incubate antibody with immunizing peptide to confirm specificity

Additionally, researchers studying NPY6R in disease contexts should consider including both normal and pathological tissues from the same origin for comparative analysis.

How does NPY6R expression correlate with cancer prognosis, particularly in uveal melanoma?

NPY6R expression demonstrates significant prognostic value in uveal melanoma (UVM):

A nomogram prediction model incorporating NPY6R expression with clinical variables (age, sex, pathologic stage, and clinical stage) demonstrates prognostic accuracy with an AUC value of 0.689 (95% CI: 0.565-0.813) for predicting one-year survival probability . This suggests NPY6R could serve as a valuable component in prognostic models for UVM patients.

What sex-specific differences in NPY6R expression have been documented in research?

Research indicates significant sex-specific differences in NPY6R expression:

Logistic regression analysis incorporating NPY6R expression with various pathological characteristics shows a significant association between gender and NPY6R expression (OR = 0.253, p = 0.004) . This suggests that:

• NPY6R expression is significantly higher in female patients compared to male patients

• The sex-specific expression pattern appears to be independent of other clinical variables

• Researchers should consider sex as an important variable when analyzing NPY6R expression data

These findings highlight the importance of sex-stratified analysis when studying NPY6R in research contexts.

How does NPY6R expression correlate with immune microenvironment characteristics?

NPY6R expression significantly correlates with multiple immune microenvironment characteristics:

• Positive correlations: NPY6R expression positively correlates with infiltration of T-helper cells, Tcm (central memory T cells), pDC (plasmacytoid dendritic cells), and CD8 T cells

• Negative correlations: NPY6R expression negatively correlates with enrichment of T cells, TFH (follicular helper T cells), NK CD56dim cells, DC (dendritic cells), cytotoxic cells, iDC (immature dendritic cells), Tem (effector memory T cells), and TReg (regulatory T cells)

• Tumor microenvironment scores: NPY6R expression significantly and negatively correlates with StromalScore, ESTIMATEScore, and ImmuneScore metrics

These correlations suggest that NPY6R may play a role in modulating the immune response within the tumor microenvironment, potentially affecting tumor progression and patient outcomes. Researchers interested in tumor immunology should consider incorporating NPY6R analysis into their experimental designs.

What methodological approaches are recommended for studying NPY6R in relation to immune cell infiltration?

When investigating NPY6R in relation to immune cell infiltration, researchers should consider the following methodological approaches:

• Computational analysis:

  • ESTIMATE algorithm: Calculate the fraction of immune cells in each patient sample

  • CIBERSORT algorithm: Determine the percentage of immune cell infiltration in tissue samples

  • Correlation analysis: Assess relationships between NPY6R expression and immune cell populations

• Laboratory techniques:

  • Multiplex immunohistochemistry: Simultaneously detect NPY6R and immune cell markers

  • Flow cytometry: Quantify immune cell populations in relation to NPY6R expression

  • Single-cell RNA sequencing: Profile expression at single-cell resolution to identify cell-specific patterns

• Validation approaches:

  • Multi-cohort analysis: Validate findings across independent patient cohorts

  • Functional studies: Assess impact of NPY6R modulation on immune cell recruitment and function

When presenting results, researchers should report both statistical significance and effect sizes, and acknowledge potential confounding factors such as tumor stage, treatment history, and patient demographics.

What pathways and biological functions are associated with NPY6R in cancer research?

Gene enrichment analyses have revealed several pathways and biological functions associated with NPY6R:

• Gene Ontology (GO) analysis: NPY6R-related genes are primarily enriched in pathways and functions related to visual light perception

• Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis: Similar to GO analysis, NPY6R-related genes show enrichment in vision-related pathways

• Gene Set Enrichment Analysis (GSEA): Suggests that NPY6R is associated with tumor progression mechanisms in uveal melanoma

• Potential mechanistic connections: Research suggests possible connections between NPY6R and the PI3K/Akt signaling pathway, which plays important roles in both retinal diseases and cancer progression

These findings indicate that NPY6R may function at the intersection of visual perception pathways and cancer biology, particularly in ocular cancers like uveal melanoma. The connection between retina-related pathways and UVM progression represents an intriguing area for further investigation.

How can researchers address contradictory findings related to NPY6R expression across different tumor types?

When faced with contradictory findings regarding NPY6R expression:

• Consider tissue specificity:

  • NPY6R expression is upregulated in some cancers (ACC, KICH, KIRC, KIRP, LAML) but downregulated in many others (BLCA, BRCA, CESC, CHOL, COAD, etc.)

  • Analyze expression patterns in context of tissue-specific functions and developmental origins

• Standardize methodological approaches:

  • Use consistent antibody clones and application protocols

  • Standardize scoring systems for immunohistochemistry

  • Employ multiple detection techniques (e.g., IHC, qPCR, Western blot) to validate expression levels

• Account for confounding variables:

  • Consider patient demographics, particularly sex-based differences

  • Document tumor stage, grade, and molecular subtypes

  • Report treatment history that may affect gene expression

• Validate with functional studies:

  • Perform gain/loss-of-function experiments to assess causality

  • Use isogenic cell line pairs to control genetic background

  • Consider three-dimensional culture models that better recapitulate in vivo conditions

By systematically addressing these considerations, researchers can better interpret seemingly contradictory findings and contribute to a more nuanced understanding of NPY6R's role in different cancer contexts.

What are the current limitations in NPY6R research and promising future directions?

Current limitations in NPY6R research include:

• Functional ambiguity: Despite being classified as a neuropeptide Y receptor, NPY6R appears unable to bind typical ligands (NPY, PP, PYY), suggesting either functional inactivity or unidentified alternative functions

• Limited mechanistic understanding: The precise mechanisms by which NPY6R influences cancer progression and immune response remain incompletely characterized

• Potential species differences: Human NPY6R may function differently from homologs in other species, complicating the translation of animal model findings

Promising future research directions include:

• Receptor-ligand identification studies: Screening for potential novel ligands that may activate NPY6R

• CRISPR-based functional genomics: Systematic gene editing approaches to elucidate NPY6R's role in cancer biology and immune modulation

• Single-cell transcriptomics: Profiling NPY6R expression at single-cell resolution to identify cell-specific functions and associations

• Sex-specific functional studies: Investigation of the mechanisms underlying observed sex differences in NPY6R expression and prognostic significance

• Therapeutic targeting potential: Evaluation of NPY6R as a potential prognostic biomarker or therapeutic target, particularly in uveal melanoma

These approaches may help overcome current knowledge gaps and potentially establish NPY6R as a clinically relevant biomarker or therapeutic target in specific cancer contexts.