NPY5R Antibody

What is NPY5R and what biological functions does it mediate?

NPY5R (Neuropeptide Y receptor type 5) functions as a receptor for neuropeptide Y and peptide YY. Its activity is mediated by G proteins that inhibit adenylate cyclase activity, with significant associations to food intake regulation and potential involvement in feeding disorders . As a member of the G protein-coupled receptor (GPCR) family, NPY5R participates in regulating energy metabolism and various other physiological processes . Recent research has also revealed its potential role as a tumor suppressor in breast cancer, where it inhibits cell growth and increases sensitivity to chemotherapeutic agents like doxorubicin .

What are the common applications for NPY5R antibodies in research?

NPY5R antibodies have demonstrated utility across multiple research applications. Western blotting represents a primary application, with validated antibodies showing detection of the approximately 50-51 kDa protein in various human tissues including brain, breast, and duodenum lysates . Immunohistochemistry applications, particularly for paraffin-embedded tissues (IHC-P), allow visualization of NPY5R in tissue contexts, with reports of specific staining in ganglion cells of human intestine . Additionally, certain antibodies such as ab133757 have been validated for immunocytochemistry/immunofluorescence (ICC/IF) and intracellular flow cytometry applications . The collective evidence from multiple manufacturers confirms cross-reactivity with both human and mouse samples .

What experimental controls should be included when using NPY5R antibodies?

When using NPY5R antibodies, researchers should implement several crucial controls. Positive controls should include tissues or cell lines with known NPY5R expression such as human hypothalamus, brain, U87 MG cells, or BxPC3 cells . Negative controls should include samples where primary antibody is omitted or replaced with isotype control antibody at equivalent concentration. For Western blotting applications, loading controls must be used to normalize protein quantities, while blocking peptides (when available) can establish binding specificity. Researchers should also consider including knockdown or knockout samples when available to definitively confirm antibody specificity to the target protein.

How should samples be prepared for optimal NPY5R detection?

Optimal NPY5R detection requires careful sample preparation tailored to the specific application. For Western blotting, protein extraction from tissues or cells should utilize lysis buffers containing protease inhibitors, with recommended loading of approximately 10 μg protein per lane . For immunohistochemistry-paraffin (IHC-P) applications, antigen retrieval is critical, with protocols recommending steamed antigen retrieval with citrate buffer at pH 6 followed by AP-staining . Immunofluorescence protocols for cultured cells like BxPC3 and U87 MG cells typically employ a 1/250 dilution of antibodies such as ab133757 . Different tissue types may require optimization of fixation methods, with formalin-fixed paraffin-embedded tissues being most commonly used for IHC.

How does NPY5R methylation affect antibody-based detection methods?

NPY5R methylation status presents important considerations for antibody-based detection strategies. Research has demonstrated significantly higher NPY5R methylation levels in breast cancer tissues compared to normal breast tissues . This epigenetic silencing mechanism directly impacts gene expression, potentially leading to reduced protein levels that may affect detection sensitivity. Researchers investigating NPY5R in cancer contexts should consider complementing protein detection with methylation analysis, as the twelve differential CpG sites located in the promoter and non-promoter regions show distinct methylation patterns between normal and tumor samples (average values of 0.22 versus 0.29, respectively) . Since antibody-based methods detect protein rather than gene silencing mechanisms, negative results should be interpreted cautiously and verified through multiple approaches.

What are the challenges in detecting NPY5R across different experimental systems?

Detecting NPY5R across experimental systems presents several technical challenges. While the predicted molecular weight is 51 kDa, observed molecular weights vary slightly between 50-51 kDa across different tissue types and detection systems . Species cross-reactivity varies by antibody clone, with some demonstrating validated reactivity only with human samples while others have confirmed reactivity with both human and mouse samples . Application compatibility also varies, with some antibodies validated only for Western blot and ELISA, while others demonstrate broader utility including flow cytometry and immunofluorescence . Researchers should carefully evaluate each antibody's validated applications and reactivity before selecting one for their specific experimental system and consider potential post-translational modifications that might affect detection.

How do NPY5R protein levels correlate with gene expression data in different tissue contexts?

The correlation between NPY5R protein levels and gene expression varies significantly across different tissue contexts. In breast cancer, both transcript and protein levels show consistent downregulation compared to normal tissues. Immunohistochemical staining confirms lower NPY5R protein levels in breast cancer tissues compared to adjacent non-tumor tissue, which aligns with quantitative PCR findings showing reduced transcript levels . Co-expression network analysis reveals that NPY5R expression strongly correlates positively with NPY1R and RBP7, while showing strong negative correlation with HM13 . These relationships suggest complex regulatory networks that may affect protein-transcript correlations in different physiological and pathological contexts. Researchers should therefore employ multiple detection methods spanning both protein and transcript levels to comprehensively characterize NPY5R status.

What signaling pathways interact with NPY5R and how might this affect experimental interpretation?

NPY5R interacts with several major signaling pathways that influence experimental interpretation. The IL6-STAT3 pathway has been implicated in NPY5R-mediated antitumor effects in breast cancer models . KEGG pathway analysis of NPY5R-coexpressed genes demonstrates enrichment in Jak-STAT, Wnt, and MAPK signaling pathways . These interactions suggest that NPY5R functions within a complex signaling network, and experimental interventions targeting NPY5R may produce effects through multiple downstream pathways. GO analysis further indicates that NPY5R-coexpressed genes are enriched in biological processes related to developmental growth, cell growth, and stem cell development . Consequently, researchers should consider these pathway interactions when designing experiments and interpreting results, particularly when evaluating NPY5R as a potential therapeutic target.

What are the optimal antibody dilutions and detection methods for different applications?

Optimal antibody dilutions and detection methods vary significantly by application and specific antibody clone. For Western blotting, recommended dilutions range from 1:1000-1:10000, with ab133757 performing optimally at 1:10000 while other antibodies like the Proteintech 30848-1-AP work best at 1:1000-1:4000 . For immunohistochemistry applications, protocols typically suggest 4-6 μg/mL concentrations for Novus antibodies and 1:250 dilutions for immunofluorescence with ab133757 . For flow cytometry applications, validation data is more limited but has been established for intracellular staining with certain antibodies . Detection methods also vary by application, with chemiluminescence commonly used for Western blotting and various fluorescence or chromogenic detection systems employed for immunostaining applications.

How can researchers optimize protocols for detecting NPY5R in different tissue samples?

Protocol optimization should start with manufacturer-recommended conditions followed by systematic adjustment of antibody concentration, incubation time, blocking reagents, and detection systems. For difficult samples, enhanced antigen retrieval methods or signal amplification systems may be necessary. Researchers should also consider tissue-specific fixation protocols, as overfixation can mask epitopes while inadequate fixation may compromise tissue morphology.

How should conflicting NPY5R expression data between different detection methods be interpreted?

• Verify antibody specificity through multiple controls including knockdown/knockout validation

• Assess mRNA integrity and potential alternative splicing that might affect detection

• Consider post-translational modifications that could mask epitopes or alter protein mobility

• Evaluate the sensitivity thresholds of different detection methods

• Analyze potential methylation or other epigenetic mechanisms affecting gene expression

Methodological triangulation using multiple antibodies and detection methods is often necessary to resolve such conflicts.

What is the significance of NPY5R in cancer research, particularly breast cancer?

NPY5R demonstrates significant relevance in cancer research, functioning as a tumor suppressor that is frequently downregulated in breast cancer . Studies analyzing TCGA, GEO databases, and independent tissue samples consistently show decreased NPY5R expression in breast cancer compared to normal tissues . The mechanism of this downregulation has been linked to promoter methylation, with significantly higher methylation levels observed in cancer tissues . Functionally, NPY5R inhibits breast cancer cell growth and increases sensitivity to doxorubicin, a common chemotherapeutic agent . This chemosensitizing effect suggests potential clinical applications for NPY5R as a biomarker or therapeutic target. The IL6-STAT3 pathway appears to mediate these antitumor effects, establishing NPY5R within a broader signaling network relevant to cancer biology .

How can researchers effectively study NPY5R methylation status?

Studying NPY5R methylation status requires specialized techniques beyond standard antibody-based detection. Researchers should:

• Utilize bisulfite sequencing to analyze the 13 CpG sites identified in the NPY5R promoter and non-promoter regions, with particular focus on the twelve sites showing differential methylation between normal and tumor samples

• Employ methylation-specific PCR for targeted analysis of specific CpG islands, particularly useful for clinical samples

• Consider methylation arrays for genome-wide analysis, which can contextualize NPY5R methylation within broader epigenetic landscapes

• Complement methylation studies with expression analysis at both transcript and protein levels to establish functional correlations

• Incorporate demethylating agents (e.g., 5-azacytidine) in experimental designs to test the functional reversibility of methylation-induced silencing

Appropriate controls should include normal tissue comparisons and cell lines with known methylation profiles.

What experimental approaches can evaluate NPY5R's role in drug sensitivity?

Evaluating NPY5R's role in drug sensitivity, particularly its reported ability to enhance doxorubicin efficacy in breast cancer , requires comprehensive experimental approaches:

• Gene modulation experiments:

  • Overexpression systems to restore NPY5R in low-expressing cancer cells

  • siRNA/shRNA knockdown in cells with normal expression

  • CRISPR-Cas9 knockout models for complete functional analysis

• Drug response assays:

  • Dose-response curves comparing NPY5R-modified versus control cells

  • Cell viability, apoptosis, and cell cycle analysis following drug treatment

  • Combination therapy assessments to identify synergistic drug partners

• Mechanistic investigations:

  • Analysis of IL6-STAT3 pathway activation through phosphorylation status

  • Assessment of drug transporter expression and function

  • Examination of apoptotic pathway components

• In vivo validation:

  • Xenograft models with NPY5R-modified cells

  • Patient-derived xenografts with varying NPY5R expression levels

  • Correlation of NPY5R status with treatment outcomes in clinical samples

These approaches can establish whether NPY5R represents a potential biomarker for chemotherapy response or a therapeutic target to enhance treatment efficacy.