NPSR1 Antibody

What is NPSR1 and why is it targeted by antibodies in research?

NPSR1 (Neuropeptide S Receptor 1) is a G-protein coupled receptor involved in the regulation of ERK1 and ERK2 signaling cascades. The canonical human NPSR1 protein has 371 amino acid residues with a molecular weight of approximately 42.7 kDa. It belongs to the G-protein coupled receptor 1 family and is primarily localized in the cell membrane and cytoplasm. Researchers target NPSR1 using antibodies as it serves as an important marker for specific neuron types, including Deep-Layer Near-Projecting Neurons, Midbrain Splatter Neurons, and Midbrain-Derived Inhibitory Neurons. Additionally, NPSR1 has been implicated in various physiological processes and pathological conditions, making it a valuable research target across multiple disciplines including neuroscience, immunology, and oncology research .

What are the common synonyms for NPSR1 that researchers should recognize in literature?

When conducting literature searches or reviewing publications, researchers should be aware of several synonyms used for NPSR1. These include G-protein coupled receptor 154, G-protein coupled receptor PGR14, G-protein coupled receptor for asthma susceptibility, vasopressin receptor-related receptor 1, and neuropeptide S receptor. Understanding these alternative nomenclatures is essential for comprehensive literature reviews and to avoid overlooking relevant research publications. The varied naming reflects the protein's discovery through different research pathways and its association with multiple physiological functions and disease states before standardization of terminology .

How many NPSR1 isoforms exist and how do they differ functionally?

Up to nine different isoforms of NPSR1 have been reported in scientific literature. The most studied isoforms are NPSR1-A and NPSR1-B, which regulate essentially identical sets of genes, though the signaling effects have been demonstrated to be stronger with NPSR1-A. These isoforms result from alternative splicing events and may exhibit tissue-specific expression patterns. The functional differences between isoforms impact downstream signaling strength and potentially receptor localization, ligand binding affinity, and physiological responses. When designing experiments with NPSR1 antibodies, researchers must consider which isoform(s) their antibodies detect to ensure proper interpretation of results .

What are the primary applications for NPSR1 antibodies in research?

NPSR1 antibodies are utilized across multiple experimental applications in research settings. Western Blot (WB) represents one of the most widely employed techniques for NPSR1 detection, allowing researchers to identify the protein and determine its molecular weight in tissue or cell lysates. Immunohistochemistry (IHC), particularly paraffin-embedded IHC (IHC-p), enables localization of NPSR1 in tissue sections, which is valuable for studies examining expression patterns in normal and disease states. Additional applications include Enzyme-Linked Immunosorbent Assay (ELISA) for quantitative analysis, Immunocytochemistry (ICC) for cellular localization studies, and Immunofluorescence (IF) for co-localization studies with other proteins. These diverse applications provide researchers with complementary approaches to investigate NPSR1 expression, localization, and function in various experimental contexts .

How should researchers optimize Western blot protocols for NPSR1 detection?

For optimal Western blot detection of NPSR1, researchers should consider several methodological factors. First, proper sample preparation is crucial—complete cell lysis with phosphatase and protease inhibitors helps preserve NPSR1 integrity, while denaturation conditions should be optimized given NPSR1's transmembrane nature. Second, gel percentage selection should accommodate NPSR1's 42.7 kDa size, with 10-12% polyacrylamide gels typically providing good resolution. Third, transfer conditions should be optimized for membrane proteins, potentially using wet transfer methods. Fourth, blocking should employ 5% BSA rather than milk to reduce background. Finally, antibody dilution requires optimization, with recommended ratios typically between 1:500-1:5000 depending on the specific antibody. Researchers should always include appropriate positive controls such as recombinant NPSR1 or lysates from cells known to express the protein, while negative controls might include knockout cell lines or tissues. Extended exposure times may be necessary as transmembrane proteins like NPSR1 can sometimes produce weaker signals than cytosolic proteins .

What methodological considerations are important for immunohistochemical detection of NPSR1?

Successful immunohistochemical detection of NPSR1 requires several important methodological considerations. Antigen retrieval is particularly critical as NPSR1 is a membrane protein and may require specific retrieval methods such as heat-induced epitope retrieval with citrate buffer (pH 6.0) or EDTA buffer (pH 9.0). Antibody concentrations should be carefully titrated, with recommended dilutions for IHC typically ranging from 1:20 to 1:200 depending on the specific antibody. Researchers should verify the epitope specificity of their NPSR1 antibodies, particularly when distinguishing between isoforms—monoclonal antibodies against the N-terminus (anti-NPSR1-N) and C-terminus (anti-NPSR1-A) may recognize different isoforms. When performing IHC on neuroendocrine tissues, parallel staining with chromogranin-A and synaptophysin is advisable to confirm neuroendocrine identity. Additionally, researchers should be aware that NPSR1 expression varies significantly across tissue types, with particularly low immunoreactivity reported in adrenal pheochromocytomas compared to other neuroendocrine tumors .

How can researchers validate the specificity of NPSR1 antibodies?

Validating NPSR1 antibody specificity requires a multi-faceted approach. First, epitope mapping should be performed using immunoblotting experiments with recombinant NPSR1 constructs expressing corresponding peptide sequences, particularly when using monoclonal antibodies targeting specific domains (N-terminus or C-terminus). Second, flow cytometry can verify binding to native NPSR1 in cell lines with known expression levels. Third, immunostaining of positive control tissues with established NPSR1 expression (such as certain regions of the skin and small intestine) should be conducted. Fourth, comparison of staining patterns between different antibodies targeting distinct epitopes of NPSR1 can confirm specificity. Fifth, preabsorption tests with immunizing peptides should abolish specific signals. Sixth, knockout/knockdown validation in cell lines using CRISPR-Cas9 or siRNA approaches provides definitive specificity confirmation. Finally, researchers should cross-reference their findings with published literature reporting NPSR1 tissue distribution. This comprehensive validation strategy ensures reliable and reproducible results in NPSR1 research .

What cross-reactivity issues should researchers be aware of when using NPSR1 antibodies?

When using NPSR1 antibodies, researchers must be vigilant about potential cross-reactivity issues that could compromise experimental results. First, sequence homology between NPSR1 and other G-protein coupled receptors may lead to non-specific binding, particularly with polyclonal antibodies. Second, antibodies raised against specific isoforms may not distinguish between all nine reported NPSR1 isoforms unless specifically designed and validated for isoform specificity. Third, cross-species reactivity should be carefully evaluated—while many NPSR1 antibodies react with human, mouse, and rat orthologs, the degree of cross-reactivity varies between antibodies and should be experimentally verified rather than assumed. Fourth, post-translational modifications like glycosylation, which has been described for NPSR1, may affect epitope accessibility and antibody binding. Fifth, fixation methods used in IHC can alter protein conformation and epitope availability. Researchers should thoroughly review validation data for their specific antibody and include appropriate controls in each experiment to account for these potential cross-reactivity issues .

How are NPSR1 antibodies used in neuroendocrine tumor research?

NPSR1 antibodies have emerged as valuable tools in neuroendocrine tumor (NET) research. They are employed to study the expression patterns of NPSR1 across different NET types, revealing that most NETs express both NPSR1 and its ligand NPS, with the notable exception of adrenal pheochromocytomas which show very low immunoreactivity. In diagnostic applications, NPSR1 immunostaining is often performed alongside established NET markers like chromogranin-A and synaptophysin to confirm neuroendocrine identity, with Ki-67 proliferation index used for tumor grading. Researchers utilize both polyclonal antibodies against NPS and monoclonal antibodies against different regions of NPSR1-A to comprehensively characterize NPS/NPSR1 expression in tumor tissues. The identification of NPSR1 in NETs has significant implications, as functional studies with NPSR1-expressing neuroblastoma cell lines demonstrate that NPS stimulation affects cancer-related pathways, including MAPK signaling, circadian activity, focal adhesion, TGF-beta signaling, and cytokine interactions. This suggests that NPS/NPSR1 signaling may influence NET biology and potentially serve as therapeutic targets .

What is the relationship between NPSR1 expression and inflammatory diseases?

NPSR1 expression has significant associations with several inflammatory diseases, making it an important research area requiring specific antibody-based detection methods. Polymorphisms in the NPSR1 gene have been linked to asthma susceptibility, potentially through alterations in receptor function or expression levels that influence inflammatory responses in respiratory tissues. Additionally, NPSR1 genetic variants have been associated with inflammatory bowel disease (IBD), suggesting a role in intestinal inflammation regulation. While no direct association between NPSR1 variants and rheumatoid arthritis (RA) diagnosis has been established, studies have identified associations of nominal significance concerning susceptibility to autoantibody-negative RA and disease activity measures. The SNP rs324987 has been specifically associated with ACPA-negative RA with an odds ratio of 0.674 (95% CI 0.512–0.888). These findings indicate that NPSR1 may play various roles in different inflammatory conditions, potentially through its influence on immune cell function, stress responses, or modulation of the neuroendocrine system. Researchers investigating these relationships require high-specificity antibodies to accurately characterize NPSR1 expression patterns in relevant tissues and cell types .

How do NPSR1 antibodies help elucidate stress-related pathways in neurological research?

NPSR1 antibodies serve as crucial tools for elucidating stress-related pathways in neurological research through multiple experimental approaches. First, they enable immunohistochemical mapping of NPSR1 distribution in brain regions associated with stress responses, particularly areas involved in the hypothalamic-pituitary-adrenal (HPA) axis. Second, using these antibodies in co-localization studies helps identify neuronal populations where NPSR1 interacts with other stress-related neurotransmitter systems. Third, NPSR1 antibodies facilitate investigation of receptor expression changes in response to various stressors, revealing adaptive mechanisms. Fourth, in combination with signaling pathway analyses, they help establish how NPS/NPSR1 signaling affects downstream targets including MAPK pathways and circadian regulation mechanisms. Fifth, these antibodies enable the study of NPSR1's role in neuroendocrine-immune interactions, particularly relevant given NPSR1's influence on both stress responses and inflammatory conditions. Research has established that NPS controls multiple neuroendocrine and behavioral responses related to stress reactions, and these antibodies are essential for dissecting the underlying molecular mechanisms through which NPSR1 modulates these complex physiological processes .

How can researchers effectively distinguish between NPSR1 isoforms using antibodies?

Distinguishing between NPSR1 isoforms requires sophisticated antibody selection and experimental design. Researchers should first select epitope-specific monoclonal antibodies targeted to regions that differ between isoforms—particularly important as NPSR1 has up to nine reported isoforms with NPSR1-A and NPSR1-B being the most studied. Antibodies targeting the C-terminus are especially useful for distinguishing NPSR1-A, as this region contains isoform-specific sequences. For complex samples containing multiple isoforms, researchers should employ isoform-specific RT-PCR in parallel with antibody-based detection to correlate protein and mRNA expression. When using Western blotting for isoform discrimination, high-resolution SDS-PAGE systems may be necessary to separate closely sized isoforms, potentially using gradient gels (4-15%) to maximize resolution. More advanced techniques like immunoprecipitation followed by mass spectrometry can provide definitive isoform identification. In functional studies, researchers should note that while NPSR1-A and NPSR1-B regulate essentially identical gene sets, signaling effects are stronger with NPSR1-A, necessitating careful interpretation of downstream effects. When reporting findings, researchers should explicitly state which isoform(s) were detected rather than making general statements about "NPSR1 expression" .

What techniques combine NPSR1 antibodies with functional assays to study signaling pathways?

Researchers investigating NPSR1 signaling pathways can employ several sophisticated techniques that combine antibody-based detection with functional assays. Phospho-specific antibody arrays can be used after NPS stimulation to simultaneously detect multiple activated signaling components, particularly within the MAPK pathway which has been identified as significantly altered following NPS/NPSR1 interaction. Single-cell immunofluorescence combined with calcium imaging allows correlation of NPSR1 expression levels with functional calcium responses to NPS stimulation on a cell-by-cell basis. For pathway dynamics, time-course immunoprecipitation experiments using NPSR1 antibodies can identify temporal changes in receptor-associated proteins following ligand stimulation. Proximity ligation assays (PLA) utilizing NPSR1 antibodies paired with antibodies against potential interacting partners can visualize protein-protein interactions in situ. CRISPR-engineered reporter cell lines expressing fluorescent-tagged NPSR1 combined with phospho-specific antibodies enable live monitoring of receptor trafficking alongside signaling activation. Transcriptome analysis of NPSR1-overexpressing cells has revealed that NPS stimulation significantly affects multiple pathways including MAPK signaling, circadian activity, focal adhesion, TGF-beta signaling, and cytokine-cytokine interactions, providing guidance for targeted investigation of these specific pathways with relevant antibody panels .

How do post-translational modifications of NPSR1 affect antibody selection and experimental design?

Post-translational modifications (PTMs) of NPSR1, particularly glycosylation, significantly impact antibody selection and experimental design in research settings. When selecting antibodies, researchers should evaluate whether the epitope region contains potential glycosylation sites, as glycosylation may mask antigenic determinants and reduce antibody binding efficiency. For studies specifically investigating NPSR1 glycosylation, antibodies targeting non-glycosylated regions are preferable for total NPSR1 detection, while lectin-binding assays can be employed in parallel to assess glycosylation status. In Western blot applications, researchers should be prepared for potential band shifts or smearing due to heterogeneous glycosylation patterns, and may need to incorporate enzymatic deglycosylation (using PNGase F or Endo H) to achieve sharper bands and confirm glycosylation status. For cell surface expression studies, it's important to note that proper glycosylation may be essential for NPSR1 trafficking to the plasma membrane, so detection protocols should be designed to distinguish between mature (fully glycosylated) and immature receptor forms. When comparing NPSR1 expression across different tissue or cell types, researchers should consider that glycosylation patterns may vary, potentially affecting antibody binding affinity and necessitating careful normalization and control experiments .

What considerations are important when using NPSR1 antibodies in multi-species comparative studies?

When conducting multi-species comparative studies using NPSR1 antibodies, researchers must address several critical considerations to ensure valid cross-species comparisons. First, sequence alignment analysis of NPSR1 orthologs (reported in mouse, rat, bovine, frog, chimpanzee, and chicken) should be performed to identify conserved epitopes that will enable consistent detection across species. Second, antibody validation should be conducted separately for each species under investigation, as cross-reactivity claimed by manufacturers may not be equivalent across all species despite sequence homology. Third, species-specific positive control samples with known NPSR1 expression should be included in all experiments. Fourth, researchers should be aware that post-translational modifications, particularly glycosylation patterns, may differ between species, potentially affecting antibody binding characteristics and resulting in species-specific band patterns on Western blots. Fifth, fixation and tissue processing protocols may require species-specific optimization to maintain epitope integrity. Sixth, when interpreting expression differences between species, researchers should consider both evolutionary divergence in NPSR1 function and potential methodological limitations in cross-species antibody reactivity. Finally, complementary molecular techniques such as species-specific PCR should be employed alongside antibody-based detection to corroborate findings across different experimental approaches .