NR1H4 Antibody

What is NR1H4 and why is it a significant research target?

NR1H4 (Nuclear Receptor Subfamily 1 Group H Member 4), also known as Farnesoid X Receptor (FXR), Bile Acid Receptor, HRR-1, RIP14, or Retinoid X receptor-interacting protein 14, is a nuclear receptor that functions as a transcription factor with a molecular mass of approximately 57 kDa . Its significance stems from its role as a global regulator of bile acid metabolism, controlling genes involved in bile acid biosynthesis (such as CYP7A1) and recycling (like IBABP) . Recent research has revealed its involvement in various cancer types, including clear cell Renal cell carcinoma (ccRCC), where it promotes cancer cell proliferation, migration, and invasion through the regulation of CCNE2 . As a nuclear receptor that binds to DNA as a heterodimer with Retinoid X Receptor (RXR), NR1H4 represents an important target for understanding both normal physiological processes and pathological conditions .

What are the key applications for NR1H4 antibodies in basic research?

NR1H4 antibodies serve multiple research applications, making them valuable tools for investigating this nuclear receptor's expression and function. The primary applications include:

• Western Blot (WB): For detecting NR1H4 protein levels in tissue or cell lysates, with expected band size at approximately 56 kDa

• Immunohistochemistry-Paraffin (IHC-P): For analyzing protein expression in fixed tissue sections

• Immunocytochemistry/Immunofluorescence (ICC/IF): For visualizing subcellular localization in cultured cells

• Flow Cytometry (Flow Cyt): For quantifying NR1H4 expression at the single-cell level

These techniques enable researchers to evaluate NR1H4 expression patterns across different tissues, subcellular compartments, and experimental conditions, providing crucial insights into its biological functions and pathological alterations.

How do I select the appropriate NR1H4 antibody for my specific research application?

Selection of an appropriate NR1H4 antibody should be based on several key considerations:

• Target epitope: Determine whether you need an antibody targeting a specific region (e.g., N-terminal as in ab187735)

• Antibody type: Consider whether monoclonal (for high specificity) or polyclonal (for broader epitope recognition) is more suitable

• Validated applications: Review validation data for your intended application (WB, IHC, ICC/IF, Flow Cytometry)

• Species reactivity: Ensure compatibility with your experimental model (human, mouse, etc.)

• Published validation: Check if the antibody has been cited in peer-reviewed publications

For instance, mouse monoclonal antibodies targeting the N-terminal region (amino acids 1-50) have been validated for multiple applications with human samples . When working with specific cell lines like A549, antibodies such as the rabbit anti-NR1H4 (A00835-1) have demonstrated effectiveness in immunofluorescence and flow cytometry applications .

What are the optimal conditions for using NR1H4 antibodies in Western blot assays?

For optimal Western blot results with NR1H4 antibodies, the following protocol parameters have been validated:

• Gel preparation: Use 5-20% SDS-PAGE gel

• Electrophoresis conditions: Run at 70V (stacking gel) followed by 90V (resolving gel) for 2-3 hours

• Sample loading: 30 μg of protein per lane under reducing conditions

• Transfer conditions: Transfer proteins to nitrocellulose membrane at 150 mA for 50-90 minutes

• Blocking: 5% non-fat milk in TBS for 1.5 hours at room temperature

• Primary antibody: Incubate with anti-NR1H4 antibody at 0.5 μg/mL overnight at 4°C

• Washing: TBS with 0.1% Tween, 3 times for 5 minutes each

• Secondary antibody: Goat anti-rabbit IgG-HRP at 1:5000 dilution for 1.5 hours at room temperature

• Detection: Use enhanced chemiluminescent detection kit

The expected molecular weight for NR1H4 is approximately 56 kDa. Different tissue samples (such as HCCT and HCCP tissue lysates) have been successfully used with this protocol .

How can I optimize immunofluorescence staining for NR1H4 localization studies?

For successful immunofluorescence detection of NR1H4, follow these methodological recommendations:

• Cell preparation: Grow cells on appropriate coverslips or chamber slides

• Fixation: Use 4% paraformaldehyde to preserve cellular structure

• Permeabilization: Apply 0.1% Triton X-100 in PBS to allow antibody access to intracellular targets

• Antigen retrieval: For some applications, perform enzyme antigen retrieval using appropriate reagents for 15 minutes

• Blocking: Block with 10% goat serum to reduce non-specific binding

• Primary antibody: Incubate with anti-NR1H4 antibody (2 μg/mL) overnight at 4°C

• Secondary antibody: Use DyLight®488 Conjugated anti-species IgG at 1:100 dilution, incubate for 30 minutes at 37°C

• Nuclear counterstain: Apply DAPI for nuclear visualization

• Visualization: Use fluorescence microscope with appropriate filter sets

This protocol has been validated with A549 cells. For co-localization studies, consider using antibodies against known interaction partners like RXR or downstream targets such as CCNE2 .

What controls should be included when performing flow cytometry with NR1H4 antibodies?

When conducting flow cytometry experiments with NR1H4 antibodies, include the following controls to ensure reliable data interpretation:

• Isotype control: Use species-matched IgG (e.g., rabbit IgG at 1 μg/10^6 cells) to assess non-specific binding

• Unstained control: Include cells without primary or secondary antibody to establish autofluorescence baseline

• Secondary-only control: Cells with secondary antibody but no primary antibody to detect non-specific secondary binding

• Positive control: Include a cell line known to express NR1H4 (e.g., A549 cells)

• Negative control: When possible, include NR1H4 knockout or knockdown cells

For intracellular staining, ensure proper fixation with 4% paraformaldehyde and permeabilization with appropriate buffer. Blocking with 10% normal goat serum is recommended. Typical antibody concentrations include primary antibody at 1 μg/10^6 cells and fluorochrome-conjugated secondary antibody at 5-10 μg/10^6 cells, with 30 minutes incubation at 20°C .

How can I establish NR1H4 knockout cell lines for functional studies?

Creating NR1H4 knockout cell lines for functional studies can be achieved through CRISPR/Cas9 technology following this methodological approach:

• Vector design: Clone CRISPR/Cas9 systems targeting NR1H4 with GFP reporter proteins

• Transfection: Transfect target cells (e.g., colon cancer cells) with the CRISPR/Cas9 construct

• Selection: After 48 hours, sort GFP-positive cells using FACS

• Single-cell cloning: Seed sorted cells onto 96-well plates at low density to obtain single-cell-derived colonies

• Clone screening: Evaluate colonies by immunoblotting for NR1H4 expression

• Validation: Verify knockout by genomic sequencing and functional assays

This approach has been successfully used to generate NR1H4 knockout colon cancer cell lines, resulting in phenotypes with impaired cell proliferation, reduced colony formation, and increased apoptotic cell death compared to control cells . Similar approaches can be adapted for other cell types based on research objectives.

What methodologies are effective for studying NR1H4's role in gene regulation?

To investigate NR1H4's role in gene regulation, several complementary methodologies can be employed:

• RT^2 Profiler PCR array: Use specialized arrays like the Human Signal Transduction Pathway Finder kit to profile altered signaling pathways in NR1H4 knockout versus wild-type cells

• Gene set enrichment analysis (GSEA): Apply this technique to identify pathways enriched in NR1H4 high versus low expression groups

• Chromatin immunoprecipitation (ChIP): Use NR1H4 antibodies to identify direct DNA binding sites

• Reporter gene assays: Construct reporters containing putative NR1H4 response elements to measure transcriptional activity

• Quantitative real-time PCR (qRT-PCR): Measure expression changes in target genes like CYP7A1 and IBABP following NR1H4 modulation

• RNA-seq: Perform transcriptome-wide analysis to identify global gene expression changes

Studies employing these approaches have revealed that NR1H4 regulates genes involved in bile acid metabolism and can also impact tumor-associated signaling pathways in cancer models .

How can I effectively study NR1H4 protein-protein interactions, particularly with RXR?

To investigate NR1H4 protein-protein interactions, especially its heterodimer formation with Retinoid X Receptor (RXR), consider these methodological approaches:

• Co-immunoprecipitation (Co-IP): Use anti-NR1H4 antibodies to pull down protein complexes, followed by immunoblotting for RXR or other potential interaction partners

• Proximity ligation assay (PLA): Detect protein interactions in situ with high sensitivity and specificity

• Fluorescence resonance energy transfer (FRET): Tag NR1H4 and potential partners with appropriate fluorophores to detect interactions in living cells

• Bimolecular fluorescence complementation (BiFC): Split fluorescent proteins fused to NR1H4 and interaction partners will reconstitute fluorescence when proteins interact

• Mammalian two-hybrid assay: Use reporter constructs to detect transcriptional activity resulting from protein-protein interactions

Since NR1H4 binds to DNA only as a heterodimer with RXR , these techniques are crucial for understanding the functional relevance of this interaction in various physiological and pathological contexts.

How should I interpret discrepancies between NR1H4 mRNA and protein expression levels in my samples?

When facing discrepancies between NR1H4 mRNA and protein expression levels, consider these methodological approaches for interpretation:

• Post-transcriptional regulation: Evaluate microRNA profiles that might target NR1H4 mRNA

• Protein stability: Assess proteasomal degradation by treating samples with proteasome inhibitors

• Translation efficiency: Perform polysome profiling to evaluate translation status

• Isoform-specific detection: Ensure that your detection methods (primers or antibodies) recognize all relevant isoforms

• Temporal dynamics: Consider time-course experiments to capture potential delays between transcription and translation

• Spatial compartmentalization: Use subcellular fractionation to determine if protein localization affects detection

Research has shown that NR1H4 expression can be complex, with bioinformatic analyses revealing multiple levels of regulation including genetic alteration and DNA methylation that significantly impact patient prognosis in cancer studies . A comprehensive multi-omics approach may be necessary to fully understand these discrepancies.

What methodological considerations are important when evaluating NR1H4 expression in cancer tissues versus normal tissues?

When comparing NR1H4 expression between cancer and normal tissues, these methodological considerations are critical:

• Tissue heterogeneity: Use laser capture microdissection to isolate specific cell populations

• Sample matching: Whenever possible, use matched tumor and adjacent normal tissue from the same patient

• Cancer subtypes: Stratify analysis by cancer subtypes, stages, and grades

• Multiple detection methods: Combine RNA-seq, qRT-PCR, immunohistochemistry, and western blotting for comprehensive assessment

• Quantification approach: For IHC, use standardized scoring systems (H-score, Allred score) and automated image analysis when possible

• Statistical analysis: Apply appropriate statistical tests and corrections for multiple comparisons

Studies have shown that NR1H4 is highly expressed in ccRCC tissues compared to normal tissues, with diagnostic potential particularly for early-stage disease (area under ROC curve > 0.8) . Similar methodological approaches can be applied to investigate NR1H4 expression in other cancer types.

How can I determine whether NR1H4 plays a causative role in cancer progression rather than being merely correlative?

To establish a causative role for NR1H4 in cancer progression beyond correlation, implement these methodological approaches:

• Genetic manipulation studies:

  • Generate NR1H4 knockout cell lines using CRISPR/Cas9

  • Create stable overexpression systems

  • Employ inducible expression systems for temporal control

• Functional assays to assess cancer hallmarks:

  • Proliferation (EdU incorporation, MTT assay)

  • Migration and invasion (transwell and wound healing assays)

  • Apoptosis (flow cytometry with Annexin V staining)

  • Colony formation assays

• Mechanistic investigations:

  • Identify downstream effectors (e.g., CCNE2)

  • Perform rescue experiments (restoring affected pathways)

  • Use pharmacological modulators of NR1H4 activity

• In vivo studies:

  • Xenograft models with NR1H4-modulated cells

  • Patient-derived xenografts

  • Genetically engineered mouse models

Research has demonstrated that NR1H4 knockdown significantly suppresses cancer cell proliferation, migration, and invasion, while mechanistic studies have identified regulation of CCNE2 as a potential mechanism in ccRCC . Similar comprehensive approaches should be applied when investigating NR1H4's role in other cancer types.

What are the best approaches for studying the relationship between NR1H4 expression and immune cell infiltration in cancer?

To investigate associations between NR1H4 and immune cell infiltration in cancer, consider these methodological approaches:

• Bioinformatic analysis:

  • Use tools like TISIDB (http://cis.hku.hk/TISIDB/index.php) to assess correlations between NR1H4 expression and tumor-infiltrating immune cells in public datasets

  • Apply deconvolution algorithms (CIBERSORT, xCell) to estimate immune cell populations from bulk RNA-seq data

• Multiplexed immunohistochemistry/immunofluorescence:

  • Perform multispectral imaging to simultaneously detect NR1H4 and immune cell markers

  • Quantify spatial relationships between NR1H4-expressing cells and immune populations

• Single-cell RNA sequencing:

  • Profile both cancer and immune cells to understand cell-specific expression patterns

  • Identify potential paracrine interactions

• Functional validation:

  • Co-culture NR1H4-modulated cancer cells with immune cells

  • Assess changes in immune cell function (cytokine production, cytotoxicity)

  • Evaluate immune checkpoint molecule expression

Research has demonstrated that NR1H4 expression is associated with immune cell infiltration levels in ccRCC, suggesting potential implications for immunotherapy approaches . These methodologies can help determine whether NR1H4 directly influences the tumor immune microenvironment or if these associations are indirect.

What strategies can address weak or absent NR1H4 signal in Western blot experiments?

When encountering weak or absent NR1H4 signal in Western blots, implement these methodological solutions:

• Sample preparation optimization:

  • Ensure complete cell lysis using appropriate buffers with protease inhibitors

  • Consider subcellular fractionation to enrich nuclear proteins

  • Optimize protein loading (50-100 μg may be needed for low abundance targets)

• Transfer conditions:

  • Extend transfer time for large proteins

  • Consider wet transfer instead of semi-dry for better efficiency

  • Verify transfer efficiency with reversible staining (Ponceau S)

• Antibody optimization:

  • Test different antibody concentrations (0.5-5 μg/mL)

  • Extend primary antibody incubation (overnight at 4°C or longer)

  • Try alternative NR1H4 antibodies targeting different epitopes

• Signal enhancement:

  • Use more sensitive detection systems (enhanced chemiluminescence)

  • Consider amplification systems for low abundance proteins

  • Reduce washing stringency if signal is completely absent

Published protocols have successfully detected NR1H4 at approximately 56 kDa using specific conditions including 5-20% SDS-PAGE gels and primary antibody concentration of 0.5 μg/mL .

How can I minimize background and optimize signal-to-noise ratio in NR1H4 immunofluorescence experiments?

To improve signal-to-noise ratio in NR1H4 immunofluorescence experiments, apply these methodological approaches:

• Fixation optimization:

  • Compare different fixatives (paraformaldehyde, methanol, acetone)

  • Minimize fixation time to prevent epitope masking

• Blocking enhancement:

  • Use species-appropriate serum (10% goat serum)

  • Add 0.1-0.3% Triton X-100 to blocking buffer

  • Consider adding 1-5% BSA to reduce non-specific binding

• Antibody conditions:

  • Titrate antibody concentration (starting at 2 μg/mL)

  • Extend primary antibody incubation time (overnight at 4°C)

  • Increase washing steps (5-6 washes of 5 minutes each)

• Advanced techniques:

  • Apply tyramide signal amplification for weak signals

  • Use confocal microscopy to reduce out-of-focus background

  • Consider spectral unmixing for autofluorescence removal

Validated protocols have successfully used anti-NR1H4 antibodies at 2 μg/mL with overnight incubation at 4°C and DyLight®488 conjugated secondary antibodies at 1:100 dilution for 30 minutes at 37°C .

What approaches can validate antibody specificity for NR1H4 in research applications?

To ensure antibody specificity for NR1H4, implement these methodological validation approaches:

• Genetic validation:

  • Test antibody in NR1H4 knockout or knockdown models

  • Compare with NR1H4 overexpression systems

  • Use siRNA-mediated silencing of NR1H4 as control

• Peptide competition:

  • Pre-incubate antibody with immunizing peptide

  • Compare staining pattern with and without peptide competition

• Multiple antibody validation:

  • Use different antibodies targeting distinct epitopes

  • Compare detection patterns across techniques (WB, IF, IHC)

• Cross-reactivity assessment:

  • Test specificity against closely related nuclear receptors

  • Perform immunoprecipitation followed by mass spectrometry

• Published validation evidence:

  • Review existing literature using the same antibody

  • Check antibody citation records in research publications

Rigorous validation is especially important for nuclear receptors like NR1H4 that may share structural similarities with other family members.