NR2F2 Antibody

What is NR2F2 and why is it important in research?

NR2F2 is an orphan nuclear receptor transcription factor that functions as a master regulator of angiogenesis and heart development. It plays vital roles in blood pressure regulation and has been implicated in multiple cancer types. Research has established NR2F2 as a genetic determinant of blood pressure, with mutations in the hinge region affecting its interaction with other proteins like Friend of GATA2 (FOG2) . Additionally, NR2F2 regulates the expression of multiple genes involved in cell cycle control, including NEK2 and RAI14, which may explain its role in cancer progression .

Methodologically, NR2F2 function can be studied using gene editing techniques (such as zinc-finger nuclease technology), RNA interference for knockdown, and chromatin immunoprecipitation (ChIP) assays to identify direct target genes.

What controls should I include when validating NR2F2 antibodies?

Proper antibody validation requires multiple controls:

• Knockout/knockdown samples: Wild-type HCT116 and NR2F2 knockout HCT116 cell lysates have been used to validate antibody specificity .

• Domain-specific controls: When studying mutant NR2F2 proteins, use antibodies targeting different domains. Research has shown that antibodies against the hinge region failed to detect mutant NR2F2 with a 5-amino acid deletion, while N-terminal antibodies successfully detected both wild-type and mutant proteins .

• Cross-reactivity controls: Test against related proteins like NR2F1 (COUP-TF I). Some antibodies (e.g., ab211777) have been validated against full-length recombinant human NR2F1 to confirm absence of cross-reactivity .

• Secondary antibody controls: Always include a secondary-only control by using PBS instead of primary antibody to identify non-specific binding .

• Tissue/cell specificity controls: Use tissues with known expression patterns. For instance, NR2F2 shows nuclear staining in mesenchymal cells of testis and lung tissues .

What are the common applications for NR2F2 antibodies?

NR2F2 antibodies have been validated for multiple research applications:

For Western blotting, the expected band size for NR2F2 is 45-46 kDa. Heat-mediated antigen retrieval with Tris/EDTA buffer pH 9.0 is recommended for immunohistochemistry applications . When analyzing tissues with mixed cell populations, separate evaluation of different compartments (epithelial vs. stromal) is essential, as alterations in compartment-specific expression have clinical significance .

How should I optimize Western blot protocols for NR2F2 detection?

Effective Western blot detection of NR2F2 requires careful optimization:

• Sample preparation: Standard RIPA buffer with protease inhibitors is suitable.

• Protein loading: 10-20 μg per lane is recommended, as demonstrated in protocols using HCT116 cell lysates .

• Gel electrophoresis: 10% SDS-PAGE gels under reducing conditions provide good separation.

• Blocking: 5% non-fat dried milk (NFDM) in TBST for 1 hour at room temperature effectively reduces background .

• Primary antibody: For monoclonal antibodies like PP-H7147-00, use 1 μg/mL; for ab211777, a 1:1000 dilution is effective .

• Incubation: Overnight at 4°C provides optimal results.

• Detection systems: Both chemiluminescence and fluorescence-based detection (IRDye-conjugated secondary antibodies) have been successfully employed .

When troubleshooting, include positive controls (cells known to express NR2F2 like MCF-7 or HCT116) and negative controls (knockdown/knockout samples). Multiple bands below the expected 45-46 kDa may represent truncated forms or cleaved fragments of NR2F2, particularly in knockout models .

What are the considerations for immunohistochemical detection of NR2F2?

Successful immunohistochemistry for NR2F2 requires specific methodological considerations:

• Antigen retrieval: Heat-mediated retrieval with Tris/EDTA buffer at pH 9.0 is critical for optimal epitope exposure .

• Antibody concentration: For ab211777, a 1:250 dilution is effective; for PP-H7147-00, 10 μg/mL is recommended .

• Detection system: HRP-conjugated secondary antibodies with DAB substrate work well, with Meyer's hematoxylin as a counterstain .

• Scoring method: Evaluate both staining intensity (0, 1+, 2+, 3+) and proportion of positive cells (0: 0-5%; 1: 6-25%; 2: 26-50%; 3: 51-75%; 4: 76-100%), examining at least 500 cells in each of 3 high-power (×40) fields .

• Compartment analysis: For tissues with heterogeneous cell populations (e.g., ovarian tissues), separately evaluate epithelial and stromal compartments. In healthy ovary, NR2F2 is predominantly stromal with minimal epithelial expression, but this pattern is disrupted in cancer .

Research has shown that ovarian cancers with the most disrupted patterns of NR2F2 localization are associated with significantly shorter disease-free intervals, highlighting the importance of proper compartment-specific analysis .

How should I approach NR2F2 knockdown experiments?

Effective NR2F2 knockdown studies require rigorous methodological controls:

• Construct selection: Both siRNA and shRNA approaches have been successful. For genetic models, Cre-mediated excision in NR2F2^f/f cells provides an alternative approach .

• Controls: Include non-targeting control (scrambled siRNA) alongside targeted knockdown. This was demonstrated in studies using siControl with siCOUP-TFII treatments .

• Validation timeline: Knockdown can be detected at 48 hours and persists for at least 6 days post-transfection .

• Multi-level confirmation: Verify knockdown at both mRNA (qRT-PCR) and protein levels (Western blot) .

• Functional validation: Monitor known NR2F2 target genes (e.g., ANF, NEK2, RAI14) to confirm functional consequences .

• Phenotypic analysis: Assess multiple parameters including proliferation and apoptosis. Research has shown that NR2F2 knockdown enhances apoptosis in serum-starved cultures of ovarian cancer cell lines and affects proliferation rates .

For comprehensive pathway analysis, RNA-seq or microarray studies following NR2F2 knockdown can identify regulated genes, with subsequent validation at protein level and ChIP experiments to determine direct binding targets.

How can I investigate NR2F2 protein-protein interactions?

The study of NR2F2 interactions requires specialized methodological approaches:

• Co-immunoprecipitation: This technique has successfully characterized the NR2F2-FOG2 interaction. Both N-terminal antibodies and tag-specific antibodies (for His-tagged NR2F2) have been employed .

• Expression vectors: Cloning NR2F2 cDNA with histidine tags into expression vectors (e.g., pCDNA 3.1(B) myc/His) enables overexpression studies and tag-based purification .

• Comparative analysis: When studying mutations, compare binding efficiency between wild-type and mutant proteins. Research has shown that a 5-amino acid deletion in the hinge region of NR2F2 enhanced its interaction with FOG2 .

• Functional validation: Use ChIP assays to determine whether altered interactions affect binding to target gene promoters. Enhanced NR2F2-FOG2 interaction led to increased binding to the ANF promoter, but not to other targets like renin and ApoB that don't require FOG2 .

• Physiological correlation: Connect molecular interactions to physiological effects. The enhanced NR2F2-FOG2 interaction correlated with reduced blood pressure, improved cardiac function, and increased vasoreactivity .

This approach has revealed that the extent of interaction between NR2F2 and FOG2 through the hinge region is linked to blood pressure regulation, providing potential therapeutic targets for hypertension.

What is the significance of NR2F2 in cancer research?

NR2F2 shows distinct expression patterns in cancer with important methodological implications:

• Expression pattern analysis: In healthy ovary, NR2F2 is robustly expressed in stroma with minimal epithelial expression. This pattern is disrupted in ovarian cancers, with decreased stromal expression and ectopic epithelial expression .

• Correlation with progression: The frequency of NR2F2 staining in epithelial compartments of metastatic ovarian cancers (≥FIGO stage II) is significantly higher than in stage I disease .

• Prognostic significance: Kaplan-Meier analyses show that ovarian cancers with the most disrupted patterns of NR2F2 are associated with significantly shorter disease-free intervals .

• Functional studies: In established ovarian cancer cell lines, NR2F2 knockdown enhances apoptosis under serum starvation and affects proliferation rates .

• Pathway analysis: NR2F2 regulates multiple genes involved in cell cycle control, including NEK2, RAI14, DCUN1D3, and SCML1, providing mechanistic insights into its role in cancer progression .

For comprehensive cancer studies, both compartment-specific immunohistochemical analysis and molecular profiling following NR2F2 modulation are recommended to fully understand its role in tumor biology.

How does NR2F2 contribute to cardiovascular regulation?

NR2F2's role in cardiovascular function has been established through several methodological approaches:

• Gene editing models: Zinc-finger nuclease technology has been used to create Nr2f2-edited rat models with a 15bp deletion in exon 2, resulting in a 5-amino acid deletion in the hinge region .

• Physiological characterization: These models show significantly lower systolic and diastolic blood pressures compared to controls .

• Cardiac function assessment: Echocardiography reveals superior left ventricular function in mutant rats, demonstrated by increased fractional shortening (56%±3 vs. 41%±2), increased velocity of circumferential shortening, and improved myocardial performance index .

• Molecular mechanism: The mutant NR2F2 protein shows enhanced binding to FOG2, with subsequent enrichment of NR2F2 binding to the ANF promoter. ANF (atrial natriuretic factor) is a known vasorelaxant .

• Functional validation: Vasorelaxation responses of mesenteric arteries from mutant rats are significantly higher than wild-type rats, confirming a functional vascular phenotype .

These findings demonstrate that NR2F2, particularly through its interaction with FOG2 mediated by the hinge region, is a genetic determinant of blood pressure with potential implications for hypertension treatment.

Why might I observe multiple bands when detecting NR2F2 by Western blot?

Multiple bands in NR2F2 Western blots may represent:

• Truncated forms/cleavage products: Bands below the expected 45-46 kDa size may represent biologically relevant fragments. Research using CRISPR/Cas9-edited cell lines has shown such bands in knockout samples .

• Post-translational modifications: As a nuclear receptor, NR2F2 may undergo modifications that alter its mobility.

• Cross-reactivity: Some antibodies may detect related family members. Test with recombinant proteins to confirm specificity.

• Splice variants: Alternative splicing can produce protein variants with different molecular weights.

To distinguish between these possibilities:

• Compare patterns using antibodies targeting different epitopes

• Use knockout/knockdown controls

• Perform peptide competition assays

• Consider subcellular fractionation to localize different forms

What factors influence NR2F2 expression in different tissues?

Understanding tissue-specific NR2F2 expression is crucial for experimental design:

• Normal expression patterns: NR2F2 is predominantly expressed in the stroma of healthy ovary, with nuclear staining in mesenchymal cells of testis and lung tissues .

• Regulatory pathways: NR2F2 expression can be modulated by Wnt signaling pathways, as demonstrated by experiments using Wnt agonists .

• Interaction with retinoic acid signaling: Studies show relationships between NR2F2 and retinoic acid receptor beta (RARβ2) transcription .

• Developmental regulation: NR2F2 expression changes during development, particularly in the context of adipogenesis and cardiovascular formation .

• Pathological alterations: In disease states like cancer, NR2F2 expression patterns become disrupted, with shifts between tissue compartments rather than simple up or down-regulation .

For developmental studies, time-course experiments are essential, while tissue-specific expression patterns can serve as internal controls for antibody specificity in immunohistochemistry.

How can I distinguish between closely related nuclear receptors in my experiments?

Distinguishing NR2F2 from related proteins requires specific methodological approaches:

• Antibody selection: Choose antibodies explicitly tested for cross-reactivity. For example, PP-H7147-00 (clone H7147) has been confirmed not to cross-react with human COUP-TF I or EAR2 .

• Epitope targeting: Select antibodies targeting regions with lower sequence conservation. While DNA binding domains are highly conserved among nuclear receptors, N-terminal regions often show greater variability.

• Validation with recombinant proteins: Test antibodies against purified recombinant NR2F1 and NR2F2 to confirm specificity, as demonstrated for ab211777 .

• Knockout controls: Use CRISPR/Cas9-edited cell lines with NR2F2 knockout as negative controls .

• Complementary approaches: Combine protein detection with mRNA analysis using primer sets specifically designed to distinguish between related transcripts.

This multi-faceted approach ensures reliable discrimination between closely related nuclear receptors, which is essential for accurate biological interpretation.