NR4A1 Antibody

What is NR4A1 and what are its alternative names in scientific literature?

NR4A1 (nuclear receptor subfamily 4 group A member 1) is a protein with several alternative designations in scientific literature, including hmr, n10, NUR77, GFRP1, NAK-1, and ST-59. This orphan nuclear receptor has a molecular weight of approximately 64.5 kilodaltons in humans . When searching literature or antibody databases, using these alternative names can ensure comprehensive coverage of relevant research resources. While primarily studied in humans, NR4A1 orthologs also exist in various model organisms including canine, monkey, mouse, and rat species, making comparative studies feasible across different animal models .

What applications are NR4A1 antibodies commonly used for in laboratory research?

NR4A1 antibodies are versatile research tools applicable across multiple experimental techniques. Common applications include:

• Western Blot (WB): For detecting NR4A1 protein expression levels in tissue or cell lysates

• Enzyme-Linked Immunosorbent Assay (ELISA): For quantitative measurement of NR4A1 in solution

• Immunohistochemistry (IHC): For visualizing NR4A1 distribution in tissue sections

• Immunofluorescence (IF): For subcellular localization studies

• Immunocytochemistry (ICC): For cellular distribution analysis

• Immunoprecipitation (IP): For studying protein-protein interactions involving NR4A1

When selecting an antibody, researchers should ensure it has been validated for their specific application. For example, antibodies from suppliers like BosterBio have been cited in publications using Western Blot applications, while products from Creative Biolabs offer broader application ranges including WB, ELISA, IF, and IP .

How can I validate the specificity of an NR4A1 antibody?

Antibody validation is critical for ensuring experimental reliability. For NR4A1 antibodies, the following validation approaches are recommended:

Immunogenic peptide blocking: This technique can confirm antibody specificity by using the immunogenic peptide (such as Ag29513) to block antibody binding. In flow cytometry experiments, when an antibody targeting the C-terminal (amino acids 299–598) of NR4A1 was validated using its immunogenic peptide in human platelets, researchers observed significant blocking of antibody binding, confirming specificity .

Permeabilization controls: When examining NR4A1 localization, compare antibody staining with and without cell permeabilization. As observed in platelet studies, antibodies targeting both N-terminal (amino acids 1–269) and C-terminal regions showed significant increases in fluorescence intensity only after permeabilization, indicating intracellular rather than surface localization of NR4A1 .

Knockout/knockdown validation: The most stringent validation utilizes genetic models lacking NR4A1 expression. In platelet-specific NR4A1-deficient mice, researchers confirmed antibody specificity by demonstrating absence of staining in knockout samples .

Multiple antibody comparison: Using antibodies recognizing different epitopes (e.g., N-terminal versus C-terminal) can provide convergent evidence for protein identification and localization.

How does NR4A1 regulate platelet activation and thrombus formation?

NR4A1 functions as a negative regulator of platelet activation and thrombus formation through a nongenomic signaling pathway. The methodological approach to study this role involves:

Genetic approaches: Generate platelet-specific NR4A1-deficient mice (Nr4a1^fl/fl^PF4-Cre) to study the impact of NR4A1 deletion specifically in platelets without confounding effects in other cell types. These models have revealed that NR4A1 deletion enhances agonist-induced platelet aggregation, integrin αIIbβ3 activation, and granule release .

In vivo thrombosis models: Three complementary models can be employed:

• FeCl3-induced carotid arterial occlusive thrombus formation

• Collagen/epinephrine-induced pulmonary thromboembolism

• Myocardial infarction models measuring microvascular microthrombi obstruction

These models collectively demonstrated that platelet-specific NR4A1 deletion accelerates thrombus formation and exacerbates pathological outcomes .

Pharmacological approaches: NR4A1-specific agonists (e.g., Csn-B) can be used to confirm the regulatory role of NR4A1. These agonists decrease platelet activation in both mouse and human platelets, providing translational evidence for NR4A1's function .

What is the role of NR4A1 in the CAP1/AC/PKA signaling pathway in platelets?

NR4A1 regulates platelet function through a nongenomic CAP1/AC/PKA signaling pathway. To investigate this mechanism:

Co-immunoprecipitation: Perform co-immunoprecipitation followed by mass spectrometry to identify NR4A1-interacting proteins. This approach revealed that adenylyl cyclase-associated protein 1 (CAP1) co-immunoprecipitated with NR4A1 in both human and mouse platelets .

cAMP measurement: Quantify 3',5'-cAMP levels in platelets after treatment with NR4A1 agonists (e.g., Csn-B) and various platelet agonists (ADP, epinephrine). Studies showed that Csn-B significantly increases cAMP levels in activated platelets and attenuates the inhibitory action of ADP on prostaglandin I2-mediated cAMP formation .

VASP phosphorylation: Monitor phosphorylation of vasodilator-stimulated phosphoprotein (VASP) at Ser157, a classical substrate of protein kinase A (PKA) in platelets. NR4A1 agonists enhance VASP phosphorylation, while NR4A1 deficiency decreases it .

This multi-parameter approach establishes the mechanistic pathway: NR4A1 binds to CAP1, which activates adenylyl cyclase, increasing cAMP levels, activating PKA, and leading to VASP phosphorylation, ultimately inhibiting platelet function .

How can I detect NR4A1 localization in platelets and does it change upon activation?

To investigate NR4A1 localization in platelets, a combinatorial approach is recommended:

Flow cytometry with selective permeabilization: Compare antibody binding with and without cell permeabilization. In human platelets, antibodies targeting both N-terminal (amino acids 1–269) and C-terminal regions (amino acids 299–598) showed increased fluorescence only after permeabilization, indicating intracellular localization .

In situ immunofluorescence microscopy: This technique provides higher resolution visualization of NR4A1 distribution within platelets. In resting human platelets, NR4A1 appears dispersed throughout the platelet cytoplasm .

Stimulation experiments: Compare NR4A1 localization in resting versus activated platelets. Interestingly, studies have shown that various physiological platelet agonists (e.g., U46619, a thromboxane A2 receptor agonist) do not cause obvious changes in NR4A1 localization in human platelets .

Subcellular fractionation: Separate platelet cytoplasmic and membrane fractions to quantitatively assess NR4A1 distribution between compartments before and after activation.

This multi-technique approach provides comprehensive information about NR4A1's localization and potential translocation during platelet activation.

How does hypercholesterolemia affect NR4A1 expression in platelets?

Hypercholesterolemia significantly impacts NR4A1 expression in platelets, requiring specific methodological approaches to investigate:

Patient and animal model selection: Compare NR4A1 expression in platelets from patients with hypercholesterolemia versus healthy controls. For animal studies, use high-fat diet (HFD)-fed mice as a hyperlipidemic model .

Expression analysis: Quantify both mRNA and protein levels of NR4A1 in isolated platelets. Studies have revealed increased expression of NR4A1 in platelets from both hyperlipidemic mice and patients with hypercholesterolemia .

Megakaryocyte investigation: Since platelets are anucleate cells derived from megakaryocytes, examine NR4A1 expression in megakaryocytes to understand the origin of increased platelet NR4A1. Research has shown that NR4A1 upregulation in platelets under hypercholesterolemic conditions derives from upregulation in megakaryocytes .

Mechanism exploration: Investigate the mechanistic basis of NR4A1 upregulation. Studies have demonstrated that hypercholesterolemia induces NR4A1 upregulation in megakaryocytes in a reactive oxygen species (ROS)-dependent manner .

This comprehensive approach reveals that hypercholesterolemia upregulates NR4A1 expression in platelets via increased expression in megakaryocytes, which is dependent on reactive oxygen species.

What are the latest approaches for targeting NR4A1 degradation in cancer research?

Recent advances in cancer research have focused on targeted protein degradation strategies for NR4A1:

Proteolysis-targeting chimera (PROTAC) development: PROTACs are bifunctional molecules that recruit an E3 ubiquitin ligase to a target protein, triggering its ubiquitination and subsequent degradation by the proteasome. Researchers have successfully developed NR-V04, a PROTAC that efficiently degrades NR4A1 in the tumor microenvironment (TME) .

Anti-tumor efficacy assessment: Evaluate the effects of NR4A1 degradation on tumor growth and progression. Studies have shown that NR-V04 has strong anti-tumor effects through targeting various pathways influenced by NR4A1 .

Tumor microenvironment analysis: Investigate how NR4A1 degradation affects the tumor microenvironment, including immune cell composition and function.

Combination therapy exploration: Assess the potential synergistic effects of combining NR4A1-degrading PROTACs with other cancer therapeutics.

This emerging approach represents a paradigm shift from traditional inhibition strategies to targeted degradation, offering new possibilities for addressing NR4A1's role in cancer.

What experimental controls are essential when studying NR4A1-protein interactions in platelets?

When investigating NR4A1 interactions with other proteins in platelets, several critical controls should be implemented:

Input controls: Always analyze a portion of the pre-immunoprecipitation lysate to confirm the presence of target proteins before pulldown.

IgG controls: Include isotype-matched IgG in parallel immunoprecipitations to identify non-specific binding.

Reciprocal immunoprecipitation: Confirm interactions by performing pulldowns in both directions (i.e., immunoprecipitate with anti-NR4A1 and probe for interacting protein, then immunoprecipitate with antibody against the interacting protein and probe for NR4A1).

Competitive blocking: Use immunogenic peptides to verify antibody specificity in the immunoprecipitation.

Treatment conditions: When studying how treatments affect interactions (e.g., NR4A1 agonist Csn-B enhancing the interaction between NR4A1 and CAP1), include appropriate vehicle controls and dose-response analyses .

Knockout/knockdown validation: Perform interactions studies in NR4A1-deficient platelets to confirm specificity of the observed interactions.

These controls collectively ensure that detected protein-protein interactions are specific and physiologically relevant.

How can I differentiate between transcriptional and nongenomic effects of NR4A1 in my research?

Distinguishing between transcriptional and nongenomic effects of NR4A1 requires specific experimental approaches:

Cell type selection: Study NR4A1 in anucleate cells like platelets where transcriptional effects are impossible. As platelets lack nuclei, any observed effects of NR4A1 must occur through nongenomic mechanisms .

Rapid timeframe analysis: Nongenomic effects typically occur within minutes, while transcriptional effects require hours. Time-course experiments can help distinguish between these mechanisms.

Subcellular localization: Track NR4A1 localization using immunofluorescence microscopy. Nuclear translocation suggests potential transcriptional activity, while cytoplasmic retention indicates nongenomic functions.

Direct protein-protein interaction studies: Use co-immunoprecipitation and mass spectrometry to identify NR4A1-interacting proteins involved in nongenomic signaling (e.g., CAP1 in platelets) .

Signaling pathway analysis: Monitor immediate downstream effects like rapid changes in second messenger levels (e.g., cAMP) and protein phosphorylation events (e.g., VASP phosphorylation) that occur too quickly to be explained by transcriptional mechanisms .

Transcription inhibitors: In nucleated cells, use transcription inhibitors (e.g., actinomycin D) or translation inhibitors (e.g., cycloheximide) to block gene expression effects and isolate nongenomic actions.

This multifaceted approach allows researchers to clearly delineate the nongenomic functions of NR4A1 from its well-established transcriptional roles.

What methodological approaches can be used to study NR4A1 as a therapeutic target in cardiovascular disease?

Investigating NR4A1 as a therapeutic target in cardiovascular disease requires integrated approaches:

Platelet function assessment: Evaluate the effects of NR4A1 agonists on platelet activation using aggregometry, flow cytometry (for integrin activation and granule release), and spreading assays. Studies have shown that NR4A1 agonists inhibit platelet activation in both healthy and hypercholesterolemic conditions .

Ex vivo and in vivo thrombosis models: Test NR4A1 agonists in various thrombosis models to assess their antithrombotic potential. Research has demonstrated that NR4A1 negatively regulates thrombus formation in multiple models .

Hypercholesterolemic models: Since NR4A1 is upregulated in platelets under hypercholesterolemic conditions, investigate whether NR4A1 agonists can normalize platelet hyperreactivity in these settings. Studies have confirmed that NR4A1 agonists significantly inhibit the activation of hypercholesterolemic platelets to the levels of control platelets .

Combined anti-atherosclerotic and antithrombotic assessment: Given that NR4A1 has been reported to have anti-atherosclerotic effects in addition to its antithrombotic activity, investigate both aspects simultaneously to develop comprehensive therapeutic approaches for atherothrombotic disease .

Drug delivery and pharmacokinetic studies: Develop and test delivery systems for NR4A1 agonists that can effectively target platelets and other relevant cardiovascular cells.

This integrated approach recognizes the multifaceted role of NR4A1 in cardiovascular pathophysiology and leverages this understanding for therapeutic development.