NR4A2 Antibody

What is NR4A2 and why are specific antibodies important for its study?

NR4A2 (also known as NURR1, NOT, IDLDP, RNR1, TINUR, and HZF-3) is a nuclear receptor transcription factor with a canonical protein length of 598 amino acid residues and a mass of 66.6 kDa in humans. It belongs to the Nuclear hormone receptor protein family and functions as a transcriptional regulator primarily localized in the nucleus and cytoplasm .

Specific antibodies are crucial for studying NR4A2 because:

• They enable detection of different isoforms (up to 2 have been reported)

• They allow differentiation between subcellular localizations

• They can be used to identify specific cell types expressing NR4A2, including Medulla Oblongata Splatter Neurons, Ventral Excitatory Neurons, and T Follicular Helper Cells

• They facilitate monitoring of expression changes during disease development or treatment

What are the major applications of NR4A2 antibodies in neuroscience research?

NR4A2 antibodies are widely used in neuroscience research due to the protein's critical role in neuronal development and function:

• Dopaminergic neuron studies: NR4A2 regulates the differentiation and maintenance of meso-diencephalic dopaminergic (mdDA) neurons during development

• Neurological disease investigations: NR4A2 mutations or dysregulation have been linked to Parkinson's disease, Alzheimer's disease, and Schizophrenia

• Developmental neurobiology: NR4A2 is involved in axon outgrowth, neuronal patterning, and terminal differentiation

• DNA repair mechanisms: NR4A2 is recruited to novel nuclear foci in response to UV irradiation and participates in nucleotide excision repair

For these applications, researchers typically employ Western blotting, immunofluorescence, and immunohistochemistry techniques with specific anti-NR4A2 antibodies.

How should I validate an NR4A2 antibody for my specific application?

Proper validation of NR4A2 antibodies is critical for experimental reliability. A comprehensive validation approach includes:

• Specificity testing:

  • Western blot analysis to confirm band at expected molecular weight (66.6 kDa)

  • Comparing staining patterns in cells known to express vs. not express NR4A2

  • Including positive controls (T-cell, B-cell, and fibroblast cell lines)

  • Using knockout/knockdown models as negative controls

• Application-specific validation:

  • For Western blotting: Optimize protein extraction (nuclear and cytoplasmic fractions)

  • For immunofluorescence: Test different fixation protocols (paraformaldehyde vs. methanol)

  • For immunohistochemistry: Compare paraffin-embedded vs. frozen sections

  • For ChIP assays: Verify with known NR4A2 binding sites (e.g., arginase 1 promoter)

• Cross-reactivity assessment:

  • Test against related NR4A family members (NR4A1, NR4A3)

  • Verify species cross-reactivity if working with non-human models (mouse, rat, etc.)

What are the optimal conditions for using NR4A2 antibodies in Western blot applications?

Based on published research protocols:

• Sample preparation:

  • Include both nuclear and cytoplasmic fractions since NR4A2 localizes to both compartments

  • Use phosphatase inhibitors if studying phosphorylation-dependent regulation

  • For tissues with potential low expression, consider immunoprecipitation prior to Western blot

• Running conditions:

  • Use 8-10% SDS-PAGE gels for optimal resolution around 66.6 kDa

  • Include positive controls from cell lines known to express NR4A2 (T-cell, B-cell lines)

• Antibody conditions:

  • Primary antibody dilutions: typically 1:1000-1:2000 (optimize for each antibody)

  • Incubation: overnight at 4°C for best results

  • Secondary antibody selection: HRP-conjugated anti-host IgG at 1:5000-1:10000

• Detection considerations:

  • Use enhanced chemiluminescence for standard detection

  • For quantitative analysis, consider fluorescent secondary antibodies and imaging

Multiple anti-NR4A2 antibodies have been successfully used in Western blot applications, including those targeting the N-terminal region (AA 13-42) , middle region, and C-terminal regions.

What protocols should be followed for immunohistochemistry with NR4A2 antibodies?

For optimal immunohistochemical detection of NR4A2:

• Tissue preparation:

  • Both paraffin-embedded and frozen sections have been successfully used

  • For paraffin sections: use heat-induced epitope retrieval (citrate buffer pH 6.0)

  • For frozen sections: fix with 4% paraformaldehyde for 10-15 minutes

• Blocking and antibody conditions:

  • Block with 5-10% normal serum (matching secondary antibody host) with 0.1-0.3% Triton X-100

  • Primary antibody incubation: 1:100-1:500 dilution overnight at 4°C

  • Secondary antibody: typically 1:200-1:500 for 1-2 hours at room temperature

• Detection systems:

  • DAB (3,3'-diaminobenzidine) for brightfield microscopy

  • Fluorescent secondary antibodies for co-localization studies

• Controls:

  • Positive tissue controls: brain sections (dopaminergic neurons), lymphoid tissues

  • Negative controls: primary antibody omission and isotype controls

• Special considerations:

  • When studying neurological tissues, perfusion fixation gives superior results

  • For inflammatory conditions, consider antigen retrieval optimization as inflammation can alter epitope accessibility

How can NR4A2 antibodies be used to study its role in cancer pathogenesis?

NR4A2 has been identified as a potential pro-oncogenic factor in certain cancers, particularly glioblastoma (GBM). Researchers can employ NR4A2 antibodies in the following advanced applications:

• Expression profiling in patient samples:

  • Immunohistochemistry and tissue microarray analysis to correlate NR4A2 expression with clinical outcomes

  • Western blot analysis of tumor vs. normal tissue samples

• Functional studies in cancer cell lines:

  • ChIP assays to identify direct transcriptional targets in cancer cells

  • Co-immunoprecipitation to detect cancer-specific protein interactions

  • Immunofluorescence for subcellular localization changes during cancer progression

• NR4A2 antagonist screening:

  • Western blot and immunofluorescence to monitor NR4A2 levels/localization after drug treatment

  • Combine with functional assays (proliferation, migration, invasion)

Research has shown that NR4A2 knockdown inhibits GBM cell and tumor growth, induces apoptosis, and inhibits migration and invasion of GBM cells. NR4A2 antagonists like DIM-C-pPhCl represent a potential new class of anti-cancer agents with applications for treating GBM .

What techniques can be used to study NR4A2 binding to DNA and its transcriptional activity?

To investigate NR4A2's function as a transcription factor:

• Chromatin Immunoprecipitation (ChIP):

  • Use ChIP-grade anti-NR4A2 antibodies (e.g., M-196 X) to immunoprecipitate DNA-protein complexes

  • Amplify bound DNA regions by PCR or sequence (ChIP-seq) to identify genome-wide binding sites

  • Known NR4A2 binding sites include the arginase 1 promoter

• Electrophoretic Mobility Shift Assay (EMSA):

  • Use recombinant NR4A2 protein or nuclear extracts

  • End-labeled DNA probes containing NR4A2 binding sites (e.g., arginase 1 promoter: 5'-GAAGTAAATGTAAGGTCAAGCGATTTTG-3')

  • Anti-NR4A2 antibodies for supershift EMSAs to confirm specificity

• Reporter gene assays:

  • Construct reporter plasmids with NR4A2 binding sites upstream of luciferase

  • Co-transfect with NR4A2 expression vectors (e.g., GAL4-Nurr1 or FLAG-Nurr1 constructs)

  • Measure transactivation by luciferase activity

  • Use in antagonist screening (e.g., DIM-C-pPhCl inhibits NR4A2-regulated transactivation)

How can NR4A2 antibodies be utilized to investigate its role in inflammation and immune regulation?

NR4A2 plays critical roles in immune cell function and inflammatory responses:

• Macrophage polarization studies:

  • Immunofluorescence and flow cytometry to detect NR4A2 in different macrophage subsets

  • Western blotting to correlate NR4A2 expression with M1/M2 markers

  • ChIP analysis to identify NR4A2 binding to promoters of M2 characteristic genes

• T-cell differentiation and function:

  • Monitor NR4A2 in regulatory T cells (Tregs) using flow cytometry

  • Co-staining with Foxp3 (NR4A2 can trans-activate Foxp3)

  • Immunoprecipitation to study protein interactions in T-cell subsets

• Inflammatory disease models:

  • Tissue immunohistochemistry in arthritis, sepsis models

  • Correlation of NR4A2 with inflammatory markers

  • Adoptive transfer experiments with NR4A2-modified immune cells

Research has shown that NR4A2 functions as a transcription factor that induces expression of M2 characteristic genes, and adoptive transfer of macrophages overexpressing NR4A2 provides protection against septic mortality . Additionally, NR4A2 is expressed at elevated levels in inflamed joint tissues from patients with rheumatoid arthritis, osteoarthritis, and psoriatic arthritis .

How should I design experiments to study NR4A2's role in cardioprotection?

Recent research has revealed NR4A2's protective role in cardiomyocytes during myocardial infarction. For investigating this function:

• In vivo experimental design:

  • Animal models: Permanent coronary ligation models

  • NR4A2 manipulation approaches:

    • Lentiviral delivery of siRNA for knockdown (lv3-siNR4A2)

    • Overexpression vectors for rescue experiments

  • Outcome measures:

    • Heart size assessment

    • Apoptosis markers (PARP, caspase3)

    • Autophagy markers (LC3)

    • Functional measurements (echocardiography)

• In vitro experimental design:

  • Cell models:

    • H9c2 cardiomyocytes with serum deprivation (mimics ischemia)

    • Neonatal rat cardiomyocytes (NRCMs) exposed to hypoxia

  • NR4A2 manipulation:

    • siRNA knockdown (verify with qPCR and western blot)

    • Overexpression plasmids for rescue

  • Assays:

    • Apoptosis detection (TUNEL staining, cleaved PARP, cleaved caspase3)

    • Autophagy assessment (LC3 conversion, using Bafilomycin A1, 3MA, rapamycin)

    • Protein interaction studies (Co-IP for NR4A2 and p53)

Data interpretation should consider that NR4A2 upregulation appears to be an adaptive response to ischemia that protects cardiomyocytes by enhancing autophagy and reducing p53/Bax-mediated apoptosis .

What controls should be included when using NR4A2 antibodies in studies of neurodevelopmental disorders?

When investigating NR4A2's role in neurodevelopmental disorders:

• Genotype controls:

  • Wild-type samples matched for age, sex, and genetic background

  • Heterozygous samples when possible (NR4A2 homozygous knockout is lethal)

  • Samples with known NR4A2 variants (e.g., missense mutations in exon 3, point mutations in exon 1)

• Tissue/cellular controls:

  • Brain region specificity (dopaminergic regions vs. control regions)

  • Cell-type controls (neurons vs. glia)

  • Developmental stage controls (embryonic, postnatal, adult)

• Technical controls for antibody validation:

  • Pre-absorption with immunizing peptide

  • Multiple antibodies targeting different epitopes

  • Knockdown validation in relevant cell types

• Domain-specific considerations:

  • Mutations in DNA binding domain (DBD) vs. ligand binding domain (LBD)

  • When studying variants, consider antibodies that recognize regions outside the mutation

Research has shown that de novo variants of NR4A2 are associated with neurodevelopmental disorders and epilepsy, with patients presenting with developmental delay, hypotonia, and seizures .

How can I reconcile contradictory data from NR4A2 studies in different inflammatory conditions?

NR4A2 exhibits context-dependent roles in inflammation, sometimes appearing pro-inflammatory and other times anti-inflammatory. To address these contradictions:

• Systematic analysis approach:

  • Compare tissue/cell types across studies (microglia vs. T cells vs. synoviocytes)

  • Examine disease models (MS vs. arthritis vs. sepsis)

  • Assess NR4A2 modifications (phosphorylation status, binding partners)

• Reconciliation strategies:

  • Perform time-course experiments (early vs. late inflammation responses)

  • Analyze all NR4A family members simultaneously (NR4A1, NR4A2, NR4A3)

  • Consider signaling context (NF-κB activity, cytokine milieu)

• Mechanistic resolution experiments:

  • ChIP-seq under different inflammatory conditions

  • Protein interaction studies in various cell types

  • Domain mutation analysis to separate different functions

Research findings show that NR4A2 can play either a proinflammatory or anti-inflammatory role depending on the immune disorder. In multiple sclerosis models, NR4A2 is selectively upregulated in T cells and augments IL-17 and IFN-γ production. In contrast, NR4A2 exerts anti-inflammatory effects by docking to NF-κB/p65 on target inflammatory gene promoters in microglia and astrocytes .

How should I design experiments to study NR4A2's role in DNA repair mechanisms?

NR4A2 has been shown to participate in nucleotide excision repair. To investigate this function:

• Subcellular localization studies:

  • Use EYFP-tagged NR4A2 to track recruitment to nuclear foci after UV irradiation

  • Co-localization with known DNA repair proteins

  • Domain analysis to identify regions required for translocation (N-terminal domain is critical)

• Signaling pathway investigation:

  • Inhibitor studies for p38 and PARP signaling (involved in NR4A2 translocation)

  • Assess impact of Ligand Binding Domain mutations

  • Test Histone Deacetylase Inhibitors to evaluate chromatin accessibility effects

• Functional DNA repair assays:

  • Measure clearance of UV-induced cyclobutane pyrimidine dimers (CPDs)

  • Measure clearance of pyrimidine-(6-4)-pyrimidone photoproduct (6-4PP) lesions

  • Compare wild-type vs. NR4A2 overexpression or knockdown conditions

Research has shown that NR4A2 over-expression facilitates more efficient clearance of UVR-induced DNA lesions, revealing a novel role for NR4A nuclear receptors as direct facilitators of nucleotide excision repair .

How can NR4A2 antibodies contribute to drug discovery for neurological disorders?

NR4A2 antibodies can facilitate drug discovery for neurological disorders through several approaches:

• Target validation strategies:

  • Immunohistochemistry to confirm NR4A2 expression in affected tissues

  • Western blot to quantify expression changes in disease states

  • ChIP to identify dysregulated transcriptional networks

• High-throughput screening applications:

  • Develop cell-based assays using NR4A2 antibodies for imaging

  • Measure nuclear translocation in response to compounds

  • Quantify protein stability/degradation after drug treatment

• Biomarker development:

  • Correlate NR4A2 levels with disease progression

  • Monitor treatment response using NR4A2 as a surrogate marker

  • Stratify patients based on NR4A2 expression patterns

Given NR4A2's essential role in dopaminergic neuron development and maintenance, compounds that modulate its activity may have therapeutic potential for Parkinson's disease, schizophrenia, and other neurological disorders .

What methodologies are appropriate for studying NR4A2 as a therapeutic target in cancer?

For investigating NR4A2 as a cancer therapeutic target:

• Target validation experiments:

  • Compare NR4A2 expression in patient-derived vs. established cancer cell lines

  • Utilize antisense oligonucleotides for selective knockdown

  • Measure effects on cell proliferation, invasion, and apoptosis markers

• Antagonist development approaches:

  • Screen compounds like DIM-C-pPhCl that act as NR4A2 antagonists

  • Assess effects on NR4A2-regulated transactivation using reporter assays

  • Confirm specificity against other NR4A family members

• In vivo efficacy evaluation:

  • Use xenograft models (e.g., U87-MG cells in athymic nude mice)

  • Measure tumor growth inhibition

  • Monitor apoptosis markers (Annexin V, caspase cleavage, PARP)

Research has demonstrated that NR4A2 is pro-oncogenic in glioblastoma, and bis-indole-derived NR4A2 antagonists represent a novel class of anti-cancer agents with potential future clinical applications .