NR2E1 Antibody

What is NR2E1/TLX and why is it important in neuroscience research?

NR2E1/TLX is a nuclear receptor that functions as a transcriptional regulator controlling the self-renewal of neural stem cells (NSCs). It has significant importance in neuroscience research because it regulates critical target genes including Pten tumor suppressor and Cdkn1a (encoding p21CIP1) . NR2E1 has been implicated as an oncogene that can initiate brain tumors, particularly glioblastomas, and is expressed strongly in neuroblastoma tissues, where its expression correlates with poor survival . The protein participates in a feedback loop with brain-specific microRNA miR-9 and interacts with the Wnt/β-catenin signaling pathway, making it a central hub in neural development and pathology research .

How should I select the appropriate anti-NR2E1 antibody for my experiment?

When selecting an anti-NR2E1 antibody, consider these methodological factors:

• Experimental application: Different antibodies perform optimally in specific applications (IHC, Western blot, ChIP, flow cytometry). For instance, when studying chromatin immunoprecipitation (ChIP) applications, select antibodies validated specifically for ChIP, as demonstrated in studies examining NR2E1 binding to the CBX7 promoter .

• Species reactivity: Ensure the antibody detects NR2E1 in your species of interest. The conservation between human and mouse NR2E1 should be considered when working with different model systems.

• Epitope location: Choose antibodies recognizing epitopes that won't be masked by protein interactions or post-translational modifications relevant to your study. This is particularly important since NR2E1 participates in multiple protein complexes.

• Validation data: Request evidence of specificity, such as knockdown controls, as shown in studies where NR2E1 was efficiently reduced using shRNA approaches .

What are the optimal fixation and permeabilization conditions for NR2E1 immunostaining?

For optimal NR2E1 immunostaining in neural tissues and cells:

• Fixation: 4% paraformaldehyde for 15-20 minutes at room temperature has been demonstrated to preserve NR2E1 antigenicity while maintaining tissue architecture. Extended fixation times may reduce antibody accessibility to nuclear antigens like NR2E1.

• Permeabilization: A brief (10-15 minute) treatment with 0.2-0.3% Triton X-100 in PBS is generally sufficient for nuclear antigen detection. For neurospheres or thicker tissues, longer permeabilization may be necessary.

• Antigen retrieval: Heat-mediated antigen retrieval (citrate buffer, pH 6.0) significantly improves detection of NR2E1 in formalin-fixed paraffin-embedded tissues, as evidenced in neuroblastoma tissue array studies .

• Blocking: Use 5-10% normal serum (from the species in which the secondary antibody was raised) with 1% BSA to minimize background staining.

How can I optimize NR2E1 antibody performance in chromatin immunoprecipitation (ChIP) experiments?

ChIP optimization for NR2E1 requires specific methodological considerations:

• Cross-linking conditions: For NR2E1 ChIP, use 1% formaldehyde for 10 minutes at room temperature. Over-fixation can mask epitopes while under-fixation may fail to preserve protein-DNA interactions.

• Sonication parameters: Aim for DNA fragments of 200-500 bp. Test sonication conditions empirically (typically 10-15 cycles of 30 seconds on/30 seconds off) and verify fragment size by agarose gel electrophoresis.

• Antibody selection: Use ChIP-validated antibodies specifically. Research has successfully employed FLAG-tagged versions of NR2E1 with anti-FLAG antibodies for enhanced specificity in ChIP experiments .

• Controls: Include:

  • Input control (non-immunoprecipitated chromatin)

  • IgG negative control (non-specific antibody)

  • Positive control (primers targeting known NR2E1 binding sites, such as the CBX7 or CDKN1A promoters)

  • Negative control regions (non-binding sites, such as PS3 as used in published protocols)

• qPCR primer design: Design primers flanking known or predicted NR2E1 binding sites. Multiple primer sets spanning different regions of the target locus can provide comprehensive binding profiles, as demonstrated in studies of NR2E1 binding to the CBX7 promoter .

Why might I observe discrepancies between NR2E1 mRNA and protein levels in my experiments?

Discrepancies between NR2E1 mRNA and protein levels can occur for several methodological and biological reasons:

• Post-transcriptional regulation: NR2E1 is regulated by microRNAs like miR-9, which can suppress translation without affecting mRNA levels . Check for microRNA expression changes in your experimental conditions.

• Protein stability regulation: NR2E1 protein may be subject to ubiquitin-mediated degradation or stabilization depending on cellular conditions. Consider measuring protein half-life using cycloheximide chase experiments.

• Feedback mechanisms: The documented feedback loop between NR2E1 and CBX7 can create complex regulatory dynamics . Time-course experiments might reveal temporal relationships between mRNA and protein levels.

• Technical considerations:

  • Antibody specificity issues (validate with knockdown controls)

  • Extraction efficiency (nuclear proteins require specialized extraction protocols)

  • Normalization methods (selection of appropriate housekeeping genes/proteins)

• Experimental factors: In developmental studies, Nr2e1 expression patterns change rapidly during embryogenesis . Precise developmental staging is critical when comparing samples.

What are the critical controls needed when using shRNA to knockdown NR2E1 expression?

When using shRNA to knockdown NR2E1, implement these methodological controls:

• Multiple shRNA sequences: Use at least two independent shRNA constructs targeting different regions of NR2E1 mRNA to confirm phenotypic effects, as demonstrated in published protocols using shNR2E1.2 and shNR2E1.3 .

• Non-targeting control: Include a non-specific shRNA (scrambled sequence) that doesn't target any known gene, such as 5'-TTCTCCGAACGTGTCACGT-3' as used in published studies .

• Empty vector control: Include the shRNA vector backbone without any inserted shRNA sequence.

• Knockdown validation: Confirm reduction of both:

  • mRNA levels (by RT-PCR, qRT-PCR)

  • Protein levels (by Western blotting)

  Aim for >80% reduction in expression for meaningful functional studies .

• Rescue experiments: Introduce an shRNA-resistant NR2E1 construct to restore expression and reverse phenotypes, confirming specificity of observed effects.

• Downstream target validation: Measure known NR2E1 targets (p21CIP1, p16INK4a, CBX7) to confirm functional consequences of knockdown .

How can I investigate the interaction between NR2E1 and the Polycomb group protein CBX7?

To investigate NR2E1-CBX7 interactions, employ these methodological approaches:

• Co-immunoprecipitation (Co-IP):

  • Perform reciprocal Co-IPs (IP with anti-NR2E1 and blot for CBX7, then IP with anti-CBX7 and blot for NR2E1)

  • Include negative controls (IgG, lysates from cells with NR2E1 or CBX7 knockdown)

  • Consider both endogenous and tagged protein approaches

• Chromatin immunoprecipitation (ChIP):

  • Perform ChIP-sequencing to identify genome-wide binding profiles

  • Conduct sequential ChIP (ChIP-reChIP) to identify regions co-occupied by both proteins

  • Use specific primer sets to examine binding at known regulatory regions, such as those used in studies demonstrating CBX7 binding at the NR2E1 locus

• Functional studies:

  • Overexpress or knockdown one factor and assess effects on the other's expression

  • Studies have shown that overexpression of CBX7 results in down-regulation of NR2E1, while knockdown of CBX7 up-regulates NR2E1

  • Evaluate effects on shared downstream targets and biological processes like senescence

• Proximity ligation assay (PLA):

  • Use PLA to visualize and quantify NR2E1-CBX7 interactions in situ

  • Compare interaction frequencies across different cell types and conditions

• Reporter gene assays:

  • Construct reporters containing NR2E1 binding sites

  • Evaluate how CBX7 modulates NR2E1-dependent transcriptional activity

What approaches can determine if NR2E1 functions differently in neural stem cells versus differentiated neural cells?

To investigate cell type-specific functions of NR2E1, implement these methodological strategies:

• Cell type-specific expression analysis:

  • Single-cell RNA-seq to compare NR2E1 expression levels across neural cell populations

  • Immunofluorescence co-staining with cell type-specific markers (Nestin for NSCs, GFAP for astrocytes, βIII-tubulin for neurons)

  • Western blot analysis of sorted cell populations

• Conditional knockdown/knockout approaches:

  • Use cell type-specific promoters to drive Cre recombinase expression

  • Compare phenotypes when NR2E1 is deleted in stem cells versus differentiated cells

  • Inducible systems to control timing of NR2E1 depletion

• ChIP-seq in different cell populations:

  • Compare NR2E1 binding sites between NSCs and differentiated neural cells

  • Integrate with histone modification data to understand chromatin context differences

  • Analyze binding motifs and co-factors that might differ between cell types

• Transcriptome analysis after manipulation:

  • RNA-seq following NR2E1 knockdown in both NSCs and differentiated cells

  • Pathway analysis to identify cell type-specific regulatory networks

  • Focus on differentiation markers, cell cycle regulators, and senescence-associated genes

• Functional assays:

  • Proliferation assays (BrdU incorporation, colony formation)

  • Differentiation potential assessment

  • Senescence markers (SA-β-Gal activity, SAHF formation)

  • Migration and invasion capabilities

How do retinoic acid (RA) treatments affect NR2E1 expression and function in neural development studies?

Retinoic acid effects on NR2E1 should be investigated using these methodological approaches:

• Dose-response and time-course experiments:

  • Test various RA concentrations (1-30 μM) and exposure times

  • Mouse studies have used 28 mg/kg RA to establish brain abnormality models

  • Monitor NR2E1 expression changes at multiple time points after treatment

• Expression analysis:

  • RT-PCR and Western blotting to quantify changes in NR2E1 mRNA and protein

  • Whole mount in situ hybridization to visualize spatial expression patterns

  • Research has demonstrated that Nr2e1 expression is significantly downregulated in the brain and NSCs of RA-treated mouse embryos

• Developmental stage considerations:

  • NR2E1 expression normally increases during normal brain development

  • RA treatment has been shown to prevent this upregulation at specific embryonic stages (E9.5 and E10.5)

  • Precise developmental staging is critical when comparing RA effects

• Mechanistic investigations:

  • ChIP assays to determine if RA receptors directly regulate the NR2E1 promoter

  • Analysis of histone modifications at the NR2E1 locus following RA treatment

  • Exploration of intermediate factors that might mediate RA effects on NR2E1

• Functional consequences:

  • Neural stem cell proliferation assays (colony counting, CCK-8 assays)

  • Analysis of Sonic Hedgehog (Shh) signaling pathway components

  • Neurosphere formation capacity (counting neurospheres with diameter >50 μm)

What are the optimal conditions for Western blot detection of NR2E1?

For optimal Western blot detection of NR2E1, follow these methodological guidelines:

• Sample preparation:

  • Nuclear extraction is critical as NR2E1 is a nuclear receptor

  • Use specialized nuclear extraction buffers containing 10-20 mM HEPES, 0.4M NaCl, 1mM EDTA, and 1mM DTT

  • Include protease inhibitors and phosphatase inhibitors freshly before use

  • Keep samples cold throughout processing

• Protein loading and transfer:

  • Load 30-50 μg of nuclear protein per lane

  • Use 8-10% polyacrylamide gels for optimal resolution

  • Transfer to PVDF membranes (preferred over nitrocellulose for nuclear proteins)

  • Transfer at lower voltage (30V) overnight at 4°C for efficient transfer of nuclear proteins

• Blocking and antibody incubation:

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

  • Dilute primary antibody in 5% BSA in TBST (typical working dilutions 1:500-1:2000)

  • Incubate with primary antibody overnight at 4°C with gentle agitation

  • Wash extensively (4 × 10 minutes) with TBST before secondary antibody

• Detection and quantification:

  • Use HRP-conjugated secondary antibodies and enhanced chemiluminescence

  • For accurate quantification, confirm signal is within the linear range

  • Normalize to nuclear loading controls (Lamin B1 or HDAC1) rather than cytoplasmic proteins like β-actin or GAPDH

• Expected molecular weight and validation:

  • Human NR2E1/TLX appears at approximately 43 kDa

  • Validate with positive controls (brain tissue) and negative controls (knockdown samples)

How can I optimize immunohistochemical detection of NR2E1 in different neural tissues?

For optimal immunohistochemical detection of NR2E1 across neural tissues:

• Tissue preparation by tissue type:

  • Fresh frozen sections: 10-12 μm thickness, acetone fixation (10 minutes at -20°C)

  • FFPE tissues: 5-7 μm sections with heat-mediated antigen retrieval (citrate buffer pH 6.0)

  • Neurospheres: 4% PFA fixation for 15 minutes, followed by sucrose cryoprotection

• Antigen retrieval optimization:

  • Compare different methods: citrate buffer (pH 6.0), EDTA buffer (pH 8.0), enzymatic retrieval

  • For neuroblastoma tissues, successful staining has been achieved with heat-mediated retrieval methods

  • Optimize time (10-30 minutes) and temperature (95-120°C)

• Background reduction strategies:

  • Pre-incubate with hydrogen peroxide (0.3% in methanol) to block endogenous peroxidases

  • Include avidin/biotin blocking step if using biotin-based detection systems

  • Add 0.1-0.3% Triton X-100 during blocking to improve antibody penetration of nuclear membranes

• Signal amplification methods:

  • Compare direct detection vs. amplification systems (ABC, tyramide)

  • For low expression tissues, tyramide signal amplification can enhance sensitivity

  • For co-localization studies, use fluorescent secondaries with different fluorophores

• Counterstaining considerations:

  • Light green counterstain has been successfully used with NR2E1/TLX immunostaining in neuroblastoma tissue arrays

  • For fluorescent detection, DAPI provides nuclear counterstaining

  • Minimize autofluorescence with Sudan Black B treatment if using fluorescent detection

What are the best approaches for quantifying NR2E1 expression in tissue microarrays?

For precise quantification of NR2E1 in tissue microarrays:

• Standardized staining protocol:

  • Process all samples simultaneously to minimize batch effects

  • Include positive and negative control tissues on each array

  • Use automated immunostaining platforms when available for consistency

• Image acquisition parameters:

  • Capture images at consistent magnification (40× has been used successfully)

  • Standardize exposure settings, white balance, and focus

  • Collect multiple fields per sample (minimum 3-5) to account for heterogeneity

• Scoring methodologies:

  • Develop a clear scoring system considering:

    • Staining intensity (0-3+)

    • Percentage of positive cells (0-100%)

    • Combined H-score (intensity × percentage, range 0-300)

  • Use both manual pathologist scoring and digital image analysis for validation

• Digital image analysis:

  • Use dedicated software with nuclear detection algorithms

  • Set consistent thresholds for positive staining across all samples

  • Validate automated quantification against manual scoring

• Statistical approaches:

  • Perform survival analysis using Kaplan-Meier methods to correlate NR2E1 expression with outcomes

  • This approach has revealed negative correlation of NR2E1 expression with survival in neuroblastoma cases

  • Consider dichotomizing expression (high vs. low) based on median or optimal cutpoint analysis

  • Adjust for multiple comparisons when necessary

How can NR2E1 antibodies be used to assess tumor stem cell populations in brain cancer specimens?

For assessment of NR2E1-positive tumor stem cells in brain cancer:

• Multi-marker immunofluorescence approach:

  • Co-stain for NR2E1 with established stem cell markers (CD133, Sox2, Nestin)

  • Include proliferation markers (Ki67) to identify actively dividing stem-like cells

  • Quantify co-expression using confocal microscopy and 3D reconstruction

• Patient-derived xenograft (PDX) models:

  • Establish PDX models from glioblastoma specimens

  • Correlate NR2E1 expression with tumor-initiating capacity and growth rates

  • Test effects of targeting NR2E1-positive cells on tumor recurrence

• Prognostic correlation analysis:

  • Quantify NR2E1 expression in large cohorts of brain tumor specimens

  • Correlate with patient outcomes and treatment response

  • Studies have shown that NR2E1 expression correlates with poor survival in neuroblastoma

• Single-cell approaches:

  • Apply single-cell RNA-seq to identify transcriptional profiles of NR2E1-high cells

  • Use flow cytometry with NR2E1 antibodies to isolate and characterize tumor subpopulations

  • Perform functional assays (sphere formation, tumorigenicity) on sorted populations

• Therapeutic response monitoring:

  • Assess changes in NR2E1-positive cell populations following treatment

  • Compare conventional therapies versus targeted approaches

  • Investigate resistance mechanisms in recurrent tumors

What are the optimal methods for studying NR2E1's role in cellular senescence?

To investigate NR2E1's involvement in cellular senescence:

• Senescence marker assessment:

  • SA-β-galactosidase activity assay (both histochemical and fluorescence-based)

  • Senescence-associated heterochromatin foci (SAHF) detection using DAPI staining

  • These markers have been successfully used to demonstrate NR2E1's role in counteracting senescence

• Experimental manipulation approaches:

  • Overexpression studies: Use retroviral vectors encoding NR2E1 to assess lifespan extension

  • Knockdown studies: Apply validated shRNAs targeting NR2E1 to induce premature senescence

  • Domain mutation analysis: Test NR2E1Δ40 mutant to establish DNA-binding dependency

• Pathway analysis:

  • Measure expression of senescence mediators (p16INK4a, p21CIP1)

  • Combine NR2E1 knockdown with p16INK4a or p21CIP1 knockdown to assess contributions

  • Quantify cumulative population doublings and colony formation capacity

• Cell type considerations:

  • Compare effects in different cell types (fibroblasts, epithelial cells, neural stem cells)

  • NR2E1 manipulation affects replicative senescence in multiple fibroblast strains (IMR90, WI-38)

  • NR2E1 knockdown also induces senescence-like responses in primary human prostate epithelial cells

• Temporal dynamics:

  • Perform time-course experiments following NR2E1 manipulation

  • Track cell proliferation (BrdU incorporation) at multiple time points

  • Monitor senescence marker emergence sequentially

How can I investigate the role of NR2E1 in the sonic hedgehog (Shh) signaling pathway?

To explore NR2E1's interactions with Shh signaling:

• Expression correlation analysis:

  • Quantify NR2E1 and Shh pathway components (Patched, Smoothened, Gli1/2/3) following NR2E1 knockdown

  • Research has demonstrated that knockdown of Nr2e1 affects the sonic hedgehog signaling pathway

  • Perform qRT-PCR, Western blotting, and immunofluorescence analyses

• Promoter binding studies:

  • Perform ChIP assays to determine if NR2E1 directly binds promoters of Shh pathway genes

  • Use reporter assays with Gli-responsive elements to assess functional impact

  • Investigate if NR2E1 interacts with Gli transcription factors using Co-IP

• Functional rescue experiments:

  • Test if activating Shh signaling (using SAG or Purmorphamine) rescues defects caused by NR2E1 knockdown

  • Conversely, determine if inhibiting Shh signaling (using cyclopamine or GANT61) prevents effects of NR2E1 overexpression

  • Assess neural stem cell proliferation and differentiation as functional readouts

• In vivo models:

  • Generate conditional knockouts of Nr2e1 in specific neural populations

  • Analyze Shh pathway activity in these models during development

  • Examine potential genetic interactions through double knockout/knockdown approaches

• Pharmaceutical modulation:

  • Screen for small molecules that affect NR2E1-Shh pathway interactions

  • Test effects on neural development and brain tumor growth

  • Investigate potential for therapeutic targeting

What techniques can be used to study NR2E1's interactions with the Wnt/β-catenin signaling pathway?

To investigate NR2E1-Wnt pathway interactions:

• Direct regulation analysis:

  • ChIP assays to confirm NR2E1 binding to Wnt7a promoter, as suggested by existing research

  • Reporter assays using Wnt7a promoter constructs with wild-type or mutated NR2E1 binding sites

  • qRT-PCR and Western blot analysis of Wnt pathway components following NR2E1 manipulation

• Functional interaction studies:

  • Test if Wnt pathway activation (using CHIR99021 or Wnt3a) can rescue defects in NSC proliferation caused by NR2E1 knockdown

  • Research has shown that Wnt/β-catenin signaling can partially rescue proliferation defects caused by NR2E1 knockdown

  • Measure TOP-flash reporter activity (β-catenin-responsive) following NR2E1 overexpression or knockdown

• Protein-protein interaction analysis:

  • Investigate potential physical interactions between NR2E1 and β-catenin using Co-IP

  • Perform proximity ligation assays (PLA) to visualize interactions in situ

  • Use domain mapping to identify interaction interfaces

• Downstream target analysis:

  • Perform RNA-seq following NR2E1 manipulation to identify shared targets with Wnt pathway

  • Compare chromatin landscapes at NR2E1 and TCF/LEF binding sites using ChIP-seq

  • Validate key targets using dual knockdown/overexpression approaches

• In vivo developmental studies:

  • Analyze expression patterns of NR2E1 and Wnt pathway components during neural development

  • Examine phenotypic consequences of manipulating both pathways simultaneously

  • Use genetic interaction studies in model organisms