NR2F6 Antibody, HRP conjugated

What is NR2F6 and why is it a significant research target?

NR2F6 (Nuclear Receptor Subfamily 2, Group F, Member 6) is a transcription factor belonging to the nuclear receptor superfamily that functions as a critical regulator in multiple biological processes. Recent research has established NR2F6 as an important molecular target due to its significant roles in:

• Cancer progression, particularly in non-small cell lung cancer (NSCLC) where it is highly expressed and correlates with invasive depth, lymphatic metastasis, and clinical staging

• Immune system regulation, including modulation of interleukin-17A production in Th17 cells

• Anti-viral immunity through its interaction with key signaling pathways including AP-1/c-Jun

• Formation of protein complexes with other regulatory factors such as HNRNPD (heterogeneous nuclear ribonucleoprotein D) that cooperatively regulate cancer progression

The multifunctional nature of NR2F6 makes it particularly relevant for oncology, immunology, and virology research, where specific antibodies are essential for detecting and quantifying its expression in various experimental systems.

How do HRP-conjugated NR2F6 antibodies differ from unconjugated versions?

HRP-conjugated NR2F6 antibodies have horseradish peroxidase enzyme directly attached to the antibody molecule, providing several methodological advantages over unconjugated versions:

Functional Differences:

• Direct detection capability without requiring secondary antibodies

• Streamlined protocols with fewer incubation and washing steps

• Enhanced sensitivity through enzymatic amplification of signal

• Reduced background from species cross-reactivity issues

• Compatibility with chromogenic substrates (TMB, DAB) and chemiluminescent detection systems

When selecting between conjugated and unconjugated antibodies, researchers should consider experimental constraints including tissue types, expected protein abundance, and detection methodologies. For dual or multiple labeling experiments, combining HRP-conjugated antibodies with antibodies bearing different conjugates (e.g., fluorophores) can allow simultaneous detection of multiple targets.

What are the validated applications for NR2F6 antibodies in research protocols?

NR2F6 antibodies have been validated for numerous experimental applications across cancer biology, immunology, and molecular biology fields:

Research has confirmed that NR2F6 antibodies are particularly valuable in studies of protein-protein interactions, as demonstrated in co-immunoprecipitation experiments verifying the direct interaction between NR2F6 and HNRNPD in lung cancer cells .

How should I optimize Western blot protocols specifically for NR2F6 detection?

Optimizing Western blot protocols for NR2F6 detection requires attention to several critical parameters:

Recommended Protocol Refinements:

• Sample Preparation:

  • Use RIPA buffer with protease inhibitors

  • Include phosphatase inhibitors if phosphorylated NR2F6 is relevant

  • Nuclear extraction protocols may increase detection sensitivity as NR2F6 is predominantly nuclear

• Gel Electrophoresis:

  • 10-12% polyacrylamide gels provide optimal resolution

  • Load 20-40 μg total protein per lane

  • Include positive control (H460 or H358 lung cancer cell lysates show high expression)

• Transfer and Blocking:

  • PVDF membranes typically outperform nitrocellulose for NR2F6 detection

  • Block with 5% non-fat milk or BSA in TBST for 1 hour at room temperature

  • For phosphorylated forms, BSA is preferable over milk

• Antibody Incubation:

  • For HRP-conjugated antibodies, start with 1:1000 dilution

  • Overnight incubation at 4°C generally yields optimal signal-to-noise ratio

  • Extended washing (5 × 5 minutes) helps minimize background

• Detection:

  • Enhanced chemiluminescence (ECL) substrates with extended sensitivity are recommended

  • Exposure times may need adjustment based on expression levels (typically 30 seconds to 5 minutes)

Researchers should also note that in lung cancer studies, NR2F6 shows differential expression between tumor and normal tissue, making careful loading control selection crucial for accurate quantification .

What controls are essential when using NR2F6 antibodies for immunohistochemistry?

Appropriate controls are critical for ensuring reliable and interpretable immunohistochemistry results with NR2F6 antibodies:

Essential Controls:

• Positive Tissue Controls:

  • Lung cancer tissue sections (particularly NSCLC) have been validated to show strong nuclear NR2F6 expression

  • Lymphoid tissues can demonstrate variable expression in immune cells

• Negative Tissue Controls:

  • Normal lung tissue typically shows minimal expression

  • Tissues known not to express NR2F6

• Antibody-Specific Controls:

  • Isotype control: Use rabbit IgG at matching concentration

  • Absorption control: Pre-incubate antibody with excess NR2F6 immunogen peptide (AA 25-53 for N-terminal antibodies)

  • Knockdown validation: Compare staining in NR2F6-knockdown versus control cells

• Technical Controls:

  • Secondary-only control (omit primary antibody)

  • Endogenous peroxidase blocking validation

  • Antigen retrieval optimization panel (citrate, EDTA, enzymatic methods)

When interpreting IHC results, researchers should note that NR2F6 expression patterns in lung cancer tissues correlate with clinical parameters including invasion depth, lymphatic metastasis, and clinical stage . Standardized scoring systems incorporating both intensity and percentage of positive cells can facilitate quantitative comparisons.

How can I validate the specificity of an NR2F6 antibody in my experimental system?

Antibody validation is essential for generating reliable research data. For NR2F6 antibodies, a comprehensive validation approach includes:

Multi-method Validation Strategy:

• Genetic Approaches:

  • Compare antibody reactivity in wild-type versus NR2F6 knockdown models

  • Research has shown clear differences in protein detection between control and shNR2F6 lentivirus-transfected H460 cells

  • Overexpression systems using tagged NR2F6 constructs

• Biochemical Validation:

  • Peptide competition assays using the immunogen peptide (e.g., AA 25-53 from N-terminal region)

  • Mass spectrometry confirmation of immunoprecipitated proteins

  • Parallel testing with multiple antibodies targeting different epitopes

• Application-Specific Verification:

  • For Western blotting: Band at expected molecular weight (~43 kDa)

  • For IHC/IF: Expected nuclear localization pattern

  • For ChIP applications: Enrichment of known NR2F6 binding sites

• Cross-reactivity Assessment:

  • Testing in species with known sequence homology

  • Evaluation in tissues with related family members (NR2F1, NR2F2)

Research has shown that NR2F6-specific EMSA (Electrophoretic Mobility Shift Assay) can be performed using probes containing the core binding sequence 5′-GTGTCAAAGGTCGTGTCAAAGGTC-3′ (with the core underlined), providing another method to validate antibody specificity through supershift assays .

How do I design co-immunoprecipitation experiments to study NR2F6 protein interactions?

Co-immunoprecipitation (Co-IP) is a powerful technique for investigating protein-protein interactions involving NR2F6, as demonstrated in studies of its interaction with HNRNPD :

Recommended Co-IP Experimental Design:

• Cell System Selection:

  • Use cell lines with documented NR2F6 expression (e.g., H460 lung cancer cells)

  • Consider creating stable cell lines expressing tagged versions (Flag-NR2F6) for enhanced detection

• Lysis Conditions:

  • Use gentle lysis buffers containing 0.5% NP-40 or 1% Triton X-100

  • Include protease/phosphatase inhibitors

  • DNase/RNase treatment may be necessary to eliminate nucleic acid-mediated interactions

• Immunoprecipitation Strategy:

  • Direct approach: Use anti-NR2F6 antibody for IP followed by blotting for potential interacting partners

  • Reverse approach: IP potential partners and blot for NR2F6

  • Tagged approach: Use anti-tag antibodies (e.g., anti-Flag) for cleaner results

• Controls:

  • IgG isotype control

  • Input sample (10% of lysate used for IP)

  • Knockdown or knockout validation

  • Reciprocal Co-IP to confirm interactions

• Detection:

  • Western blotting with specific antibodies for predicted interacting proteins

  • Mass spectrometry for unbiased identification of novel binding partners

This approach has successfully demonstrated the physical interaction between NR2F6 and HNRNPD proteins in H460 lung cancer cells. Flag-tagged NR2F6 was shown to co-precipitate with HNRNPD, and reciprocally, Flag-tagged HNRNPD co-precipitated with NR2F6, confirming their interaction .

What experimental approaches can elucidate NR2F6's role in transcriptional regulation?

NR2F6 functions as a transcription factor with diverse regulatory roles. Multiple complementary approaches can be employed to investigate its transcriptional functions:

Transcriptional Regulation Analysis Methods:

• Chromatin Immunoprecipitation (ChIP):

  • Use NR2F6 antibodies validated for ChIP applications

  • Analyze enrichment at predicted binding sites

  • Follow with qPCR for targeted analysis or sequencing for genome-wide profiling

  • NR2F6 binding elements often contain the core sequence found in COUP-TF family members

• Reporter Gene Assays:

  • Construct luciferase reporters containing putative NR2F6-responsive elements

  • Compare reporter activity in NR2F6 knockdown/overexpression versus control conditions

  • Include mutated binding site controls

• Gene Expression Analysis:

  • Compare transcriptomes in NR2F6 knockdown versus control cells

  • RNA-Seq analysis has identified NR2F6-regulated gene networks in innate immune pathways

  • Validate key targets with RT-qPCR and protein analysis

• Protein-DNA Interaction Studies:

  • Electrophoretic Mobility Shift Assay (EMSA) with the NR2F6 binding sequence

  • Use supershift assays with NR2F6-specific antibodies to confirm specificity

  • DNA affinity precipitation assays for protein complex identification

Research has demonstrated that NR2F6 can bind to the promoter of MAP3K5 and activate the AP-1/c-Jun pathway, which is critical for DNA virus replication . Additionally, NR2F6 itself appears to be transcriptionally regulated through a negative feedback loop involving c-Jun and the cGAS/STING pathway through STAT3 .

How can I interpret contradictory results between NR2F6 RNA and protein expression levels?

Discrepancies between RNA and protein levels of NR2F6 are not uncommon and require careful interpretation:

Analysis Framework for Resolving Discrepancies:

• Methodological Considerations:

  • RNA detection methods (RT-qPCR, RNA-Seq) measure steady-state mRNA levels

  • Protein detection methods (Western blot, IHC) capture post-transcriptional regulation effects

  • Antibody epitope accessibility may be affected by protein modifications or interactions

• Biological Explanations:

  • Post-transcriptional regulation (miRNAs, RNA binding proteins)

  • Post-translational modifications affecting protein stability

  • Protein-protein interactions altering antibody recognition

  • Subcellular localization changes affecting detection in certain fractions

• Analytical Approach:

  • Temporal analysis: Measure RNA and protein at multiple time points

  • Fractionation studies: Analyze nuclear versus cytoplasmic compartments

  • Protein stability assessment: Cycloheximide chase experiments

  • Proteasome inhibition: MG132 treatment to assess degradation pathways

• Validation Strategies:

  • Use multiple antibodies targeting different epitopes

  • Employ alternative detection methods (mass spectrometry)

  • Create tagged-NR2F6 expression systems for orthogonal validation

Research has shown that NR2F6 regulation occurs at multiple levels. For example, in viral infection contexts, cGAS/STING innate immunity signaling represses NR2F6 expression through STAT3, while c-Jun forms a negative feedback loop to control NR2F6 levels . These complex regulatory mechanisms may explain observed discrepancies between RNA and protein measurements.

What are the key considerations for studying NR2F6 in the context of cancer immunology?

NR2F6 has emerging roles at the intersection of cancer and immunology, requiring specialized experimental approaches:

Cancer Immunology Research Considerations:

• Tumor Microenvironment Analysis:

  • Multiplex immunofluorescence to co-localize NR2F6 with immune cell markers

  • Single-cell RNA-Seq to define NR2F6 expression in specific immune populations

  • Spatial transcriptomics to map NR2F6 expression patterns within tumor regions

• Immune Function Assessment:

  • T cell activation assays in the context of NR2F6 modulation

  • Cytokine production profiles (particularly IL-17A)

  • Immune cell migration and infiltration studies

• Functional Interrogation:

  • Conditional knockout models specific to immune cell populations

  • Adoptive transfer models to assess NR2F6-modified immune cells in tumor control

  • In vitro co-culture systems with tumor and immune cells

• Therapeutic Implications:

  • Combination studies with immune checkpoint inhibitors

  • Effects of NR2F6 targeting on conventional cancer therapies

  • Biomarker development for stratifying immunotherapy responders

NR2F6 has been shown to directly antagonize NFAT and regulate IL-17A production in Th17 cells , suggesting it may play important roles in modulating anti-tumor immune responses. The functional connections between NR2F6's roles in cancer progression and immune regulation make it a particularly interesting target at the cancer-immunology interface.

How can I optimize ChIP-seq protocols for studying NR2F6 interactions with DNA?

ChIP-seq for NR2F6 requires careful optimization to capture its DNA binding profile accurately:

ChIP-seq Optimization Strategy:

• Cross-linking Optimization:

  • Test multiple formaldehyde concentrations (0.5-2%)

  • Consider dual cross-linking with DSG or EGS for more stable protein-protein interactions

  • Optimize cross-linking times (5-20 minutes)

• Chromatin Preparation:

  • Compare sonication versus enzymatic digestion methods

  • Verify fragment size distribution (aim for 150-300 bp)

  • Ensure consistent chromatin input amounts across samples

• Antibody Selection and Validation:

  • Use ChIP-grade NR2F6 antibodies specifically validated for this application

  • Perform preliminary ChIP-qPCR at known or predicted binding sites

  • Include isotype control antibodies as negative controls

• IP Optimization:

  • Test different antibody amounts (2-10 μg per reaction)

  • Optimize bead type and amount

  • Adjust washing stringency to balance specificity and yield

• Library Preparation and Sequencing:

  • Include input controls for normalization

  • Consider spike-in controls for quantitative analyses

  • Use sufficient sequencing depth (>20 million reads)

• Data Analysis:

  • Focus on motif analysis for the NR2F6 binding sequence, similar to that used in EMSA studies (5′-GTGTCAAAGGTCGTGTCAAAGGTC-3′)

  • Integrate with RNA-seq data to correlate binding with transcriptional outcomes

  • Perform differential binding analysis across experimental conditions

H3K27ac ChIP-Seq analysis has been successfully used to identify NR2F6 as an important host factor involved in the signaling network activated by viral infection , demonstrating the utility of this approach for understanding NR2F6 function in different biological contexts.

What are the most reliable methods for quantifying NR2F6 protein expression in tissue samples?

Accurate quantification of NR2F6 in tissues requires consideration of several methodological approaches:

Quantification Methods Comparison:

For NR2F6 quantification in lung cancer studies, immunohistochemistry has provided valuable insights into expression patterns that correlate with clinical parameters . When performing IHC quantification, H-score methods that combine staining intensity and percentage of positive cells have proven effective for correlating NR2F6 expression with clinicopathological features and prognosis.

How can I effectively study the functional impact of NR2F6 in cellular models?

Investigating NR2F6's functional roles requires systematic approaches to manipulate its expression and activity:

Functional Analysis Framework:

• Expression Modulation:

  • RNA interference: siRNA or shRNA for transient or stable knockdown

  • CRISPR-Cas9: Gene knockout or mutation of specific domains

  • Overexpression: Wild-type or mutant constructs

• Phenotypic Assays:

  • Proliferation: CCK8 assay has demonstrated reduced proliferation in NR2F6 knockdown lung cancer cells

  • Colony formation: Significantly reduced in NR2F6 knockdown models

  • Migration and invasion: Transwell assays to assess metastatic potential

  • Apoptosis: Flow cytometry with Annexin V/PI staining

• Pathway Analysis:

  • Western blotting for downstream effectors

  • Phospho-protein arrays to identify signaling changes

  • Reporter assays for pathway-specific activation

• Target Validation:

  • Rescue experiments with wild-type or mutant constructs

  • Combinatorial knockdown/overexpression with interacting partners

  • Pharmacological pathway modulation to confirm mechanisms

• Physiological Relevance:

  • 3D culture models to better recapitulate in vivo conditions

  • Organoid systems for tissue-specific effects

  • Xenograft models for in vivo validation

Research has demonstrated that NR2F6 knockdown significantly inhibits proliferation and colony formation in H460 and H358 lung cancer cell lines . Similarly, knockdown of HNRNPD, which interacts with NR2F6, produces comparable effects, suggesting they function cooperatively in regulating cancer cell growth .

What are the latest findings regarding NR2F6's role in cancer progression and potential as a therapeutic target?

Recent research has revealed several important aspects of NR2F6 biology with therapeutic implications:

Current Understanding:

NR2F6 has emerged as a significant factor in cancer progression, particularly in non-small cell lung cancer (NSCLC). Key findings include:

• High expression of NR2F6 in lung cancer tissues correlates with poor prognosis and aggressive clinicopathological features

• NR2F6 knockdown significantly inhibits proliferation of lung cancer cells, suggesting its potential as a therapeutic target

• Interaction between NR2F6 and HNRNPD appears to jointly regulate cancer progression, highlighting the importance of protein-protein interactions in NR2F6's oncogenic functions

• NR2F6 involvement in immune regulation suggests potential impacts on tumor immunosurveillance

Therapeutic Targeting Approaches:

Based on these findings, several therapeutic strategies targeting NR2F6 are being explored:

• Direct inhibition of NR2F6 expression or function

• Disruption of critical protein-protein interactions (e.g., NR2F6-HNRNPD)

• Combination approaches targeting NR2F6 alongside immune checkpoint inhibitors

• Biomarker development for patient stratification based on NR2F6 expression levels

These emerging directions suggest that NR2F6-targeting approaches may hold promise for cancer therapy, particularly in NSCLC where high expression correlates with poor clinical outcomes .

How does NR2F6 function in the context of viral infections and host immune responses?

NR2F6 plays complex roles in viral infections and host immune responses:

Viral Infection Context:

Recent studies have revealed that NR2F6 significantly impacts host-virus interactions:

• NR2F6 promotes HSV-1 (herpes simplex virus) replication and gene expression both in vitro and in vivo

• NR2F6 binds to the promoter of MAP3K5 and activates the AP-1/c-Jun pathway, which is critical for DNA virus replication

• A negative feedback loop exists where NR2F6 is transcriptionally repressed by c-Jun

• The cGAS/STING innate immunity signaling pathway represses NR2F6 expression through STAT3, creating another regulatory layer

Immune Response Regulation:

Beyond viral contexts, NR2F6 functions as an important immune regulator:

• NR2F6 directly antagonizes NFAT and regulates IL-17A production in Th17 cells

• NR2F6 modulation affects T cell activation and function in autoimmune disease models

• RNA-Seq analysis of NR2F6 knockdown cells reveals regulation of innate immune pathways

These findings position NR2F6 at a critical intersection between viral infection, innate immunity, and potentially tumor immunosurveillance, suggesting it may be an important integrator of these interconnected biological systems.

The complexity of NR2F6 function across multiple biological contexts highlights its potential as both a therapeutic target and a fundamental research subject for understanding the interplay between cancer, immunity, and infection.