NR3C2 Antibody

What is NR3C2 and why is it significant in research?

NR3C2 (Nuclear Receptor Subfamily 3, Group C, Member 2) encodes the mineralocorticoid receptor (MR), which mediates aldosterone actions on salt and water balance within restricted target cells. The protein functions as a ligand-dependent transcription factor that binds to mineralocorticoid response elements to transactivate target genes. Recent research has identified NR3C2 as a tumor suppressor in various cancers, making it an important research target. It has a molecular weight of approximately 107 kDa and may also be known as MCR, MR, MLR, or aldosterone receptor .

What should researchers consider when selecting an NR3C2 antibody?

When selecting an NR3C2 antibody, researchers should consider:

• Target specificity: Verify the exact epitope region (N-terminal, middle region, or C-terminal)

• Validated applications: Ensure the antibody has been validated for your specific application (WB, IHC, IF, etc.)

• Species reactivity: Confirm cross-reactivity with your study species (human, mouse, rat, etc.)

• Clonality: Choose between monoclonal (higher specificity) or polyclonal (broader epitope recognition)

• Validation data: Review published studies using the antibody

• Immunogen information: Check whether it was raised against a synthetic peptide, recombinant protein, or using anti-idiotypic methods

How do I determine the appropriate NR3C2 antibody dilution for my experiments?

Optimal antibody dilutions vary by application:

• Western Blotting: Most NR3C2 antibodies work at dilutions between 1:500-1:8000

• Immunohistochemistry: Typically 1:50-1:500

• Immunofluorescence: Usually 1:50-1:800

Always perform a dilution series to determine optimal conditions for your specific sample and protocol. Many antibodies show effective signals at 1:1000 for WB and 1:200 for IHC/IF applications. Due to the relatively low expression of NR3C2 in many tissues, enhanced detection systems are recommended for Western blotting, such as enhanced chemiluminescence .

What are the validated applications for NR3C2 antibodies?

NR3C2 antibodies have been validated for multiple applications:

Note that some antibodies have specific limitations; for example, the MA1-620 monoclonal antibody cannot be used for immunoprecipitation .

How should I prepare samples for NR3C2 detection in different tissue types?

Sample preparation varies by tissue and detection method:

For protein extraction:

• Use RIPA lysis buffer with protease inhibitor PMSF

• Separate by 10% SDS-PAGE

• Transfer to polyvinylidene difluoride membranes

For IHC in formalin-fixed paraffin-embedded samples:

• Perform antigen retrieval using TE buffer (pH 9.0) or citrate buffer (pH 6.0)

• Block endogenous peroxidase activity

• Use appropriate positive controls (kidney tissues show high NR3C2 expression)

Different tissues express varying levels of NR3C2, with highest expression in kidney, colon, and certain brain regions. Heart tissue shows moderate expression with localization in myocytes and endothelial cells .

What positive controls should I use for validating NR3C2 antibody performance?

Recommended positive controls for NR3C2 antibody validation:

• Tissues: Kidney tissue (human or mouse), colon tissue

• Cell lines: HEK-293 cells, HeLa cells

• Overexpression systems: Cells transfected with NR3C2 expression vectors

Western blotting typically shows bands at 94-110 kDa, corresponding to the predicted molecular weight of NR3C2. Multiple bands may indicate splice variants or post-translational modifications .

How does NR3C2 expression differ between normal tissues and cancer tissues?

Multiple studies have demonstrated that NR3C2 is downregulated in various cancer types:

Immunohistochemistry staining and western blotting analyses consistently confirm the downregulation of NR3C2 at both mRNA and protein levels in cancer tissues. This pattern suggests NR3C2 functions as a tumor suppressor gene across multiple cancer types .

What are the molecular mechanisms of NR3C2's tumor suppressor function?

NR3C2 exhibits tumor suppressor activity through several mechanisms:

• Inhibition of the AKT/ERK signaling pathway:

  • Overexpression of NR3C2 significantly reduces phosphorylation of AKT and ERK

  • This inhibits downstream effectors of cancer cell proliferation and survival

• Suppression of angiogenesis:

  • Decreases expression of phosphorylated VEGF receptor 2

  • Reduces VEGF levels and inhibits tube formation

• Inhibition of invasion and migration:

  • Decreases expression of matrix metalloproteinases (MMP2 and MMP9)

  • Suppresses cell motility and invasive capabilities

• Modulation of the tumor immune microenvironment:

  • Higher NR3C2 expression correlates with increased immune and stromal scores

  • Positive correlation with infiltration of CD4+ and CD8+ T cells

How can NR3C2 be used as a prognostic biomarker in cancer research?

NR3C2 shows significant potential as a prognostic biomarker:

These findings suggest NR3C2 status could be incorporated into prognostic models for multiple cancer types .

How can genetic and epigenetic regulation of NR3C2 be studied using antibody-based approaches?

To investigate genetic and epigenetic regulation of NR3C2:

• Copy Number Variation (CNV) analysis combined with protein expression:

  • Correlate CNV data with protein expression using western blot

  • NR3C2 antibodies can help quantify protein levels in samples with known CNV status (deletion, normal, or amplification)

  • In NSCLC studies, CNV was classified based on copy numbers: -1 or -2 (deletion), 0 (normal), 1 or 2 (amplification)

• Chromatin Immunoprecipitation (ChIP) for histone modification analysis:

  • Use NR3C2 antibodies with ChIP protocols to assess protein-DNA interactions

  • Combine with histone marker antibodies to investigate epigenetic regulation

• Methylation-protein expression correlation:

  • In LUSC, promoter methylation at specific sites correlates with decreased protein expression

  • Antibody-based detection of NR3C2 can be paired with bisulfite sequencing data

  • Two NR3C2 promoter methylation sites showed high sensitivity and specificity for LUSC diagnosis

How should researchers approach NR3C2 antibody-based co-immunoprecipitation experiments to study protein interactions?

For effective co-immunoprecipitation of NR3C2 interacting proteins:

• Select appropriate antibody:

  • Use antibodies specifically validated for immunoprecipitation

  • Typically 0.5-4.0 μg antibody per 1.0-3.0 mg of total protein lysate

  • Note that some antibodies (e.g., MA1-620) are not suitable for IP

• Optimize lysis conditions:

  • For nuclear receptors like NR3C2, nuclear extraction protocols are crucial

  • Consider non-denaturing conditions to preserve protein-protein interactions

• Confirm binding partner candidates:

  • NR3C2 interacts with heat shock proteins (Hsp90) and High Mobility Group Box (HMGB) proteins

  • Can form heterodimers with other nuclear receptors, particularly glucocorticoid receptor (NR3C1)

• Validate interactions using reciprocal IP:

  • Immunoprecipitate with antibodies against suspected binding partners

  • Blot with NR3C2 antibody to confirm interaction

What considerations are important when studying NR3C2 in tumor microenvironment (TME) studies?

For TME studies involving NR3C2:

• Multi-parameter immunofluorescence approach:

  • NR3C2 antibodies can be combined with immune cell markers (CD4, CD8, etc.)

  • Evaluate co-localization patterns using confocal microscopy

  • Research shows NR3C2 expression positively correlates with infiltration of B cells, CD8+ T cells, CD4+ T cells, macrophages, neutrophils, and dendritic cells

• Tissue microarray (TMA) analysis:

  • Use NR3C2 antibodies optimized for IHC on TMAs containing tumor and adjacent tissues

  • Score expression levels and correlate with clinical outcomes and immune markers

  • Significant associations between NR3C2 levels and stromal/immune scores have been reported

• Ex vivo culture systems:

  • Study NR3C2 expression in tumor spheroids co-cultured with immune cells

  • Use immunofluorescence to track NR3C2 expression changes in response to immune cell interaction

Research has shown that lower NR3C2 expression correlates with reduced immune scores, stromal scores, and ESTIMATE scores in multiple cancer types, suggesting its importance in modulating the tumor microenvironment .

What are the common challenges in detecting NR3C2 and how can researchers overcome them?

Common challenges and solutions:

• Low endogenous expression levels:

  • Use enhanced detection systems (ECL Plus) for Western blotting

  • Employ signal amplification methods for IHC/IF

  • Consider concentrating proteins when preparing samples

  • Due to extremely low NR3C2 levels in native tissues, enhanced chemiluminescence detection is recommended

• Non-specific binding:

  • Optimize blocking conditions (5% BSA often works better than milk for nuclear receptors)

  • Include additional washing steps

  • Use monoclonal antibodies for higher specificity applications

• Splice variant detection:

  • Be aware that NR3C2 has multiple isoforms (observed MW ranges from 94-110 kDa)

  • Use antibodies targeting conserved regions to detect all isoforms

  • For specific isoform detection, select antibodies targeting unique regions

• Cross-reactivity with other steroid receptors:

  • Some antibodies (e.g., MA1-620) may cross-react with other receptors with similar steroid binding domains

  • Validate specificity using knockout/knockdown controls

  • Consider competitive binding assays (MA1-620 has been shown to block aldosterone binding)

How can researchers validate the specificity of their NR3C2 antibody results?

To validate antibody specificity:

• Genetic validation:

  • Use NR3C2 knockdown or knockout samples as negative controls

  • Published studies have utilized NR3C2 shRNA for validation (sequence: CCGG-CCAGCTAAGATTTATCAGAAT-CTCGAG-ATTCTGATAAATCTTAGCTGG-TTTTTT)

• Peptide competition assays:

  • Pre-incubate antibody with immunizing peptide before application

  • Signal should be significantly reduced or eliminated

  • For MA1-620, pre-incubation with aldosterone blocks staining

• Multiple antibody validation:

  • Use antibodies from different sources targeting different epitopes

  • Consistent results across antibodies increase confidence in specificity

• Recombinant protein controls:

  • Compare signal with samples containing overexpressed NR3C2

  • Use GFP-tagged NR3C2 to confirm localization in IF/ICC experiments

What are the considerations for interpreting NR3C2 expression patterns in different subcellular compartments?

Interpreting subcellular localization:

• Expected localization patterns:

  • NR3C2 is primarily detected in the cytoplasm in inactive state

  • Upon ligand binding, it translocates to the nucleus

  • Some cells show both nuclear and cytoplasmic localization

  • NR3C2 subcellular location is listed as cytoplasm, nucleus, and cell membrane

• Interpretation in cancer studies:

  • Altered subcellular localization may indicate dysregulation

  • Nuclear exclusion could suggest impaired function as a transcription factor

  • Quantify nuclear/cytoplasmic ratio for more objective assessment

• Technical considerations:

  • Fixation methods can affect observed localization

  • Optimize permeabilization protocols for nuclear detection

  • Use proper subcellular markers (nuclear, cytoplasmic) as co-staining controls

• Aldosterone stimulation:

  • Consider examining NR3C2 localization with and without aldosterone stimulation

  • This can help differentiate between functional and non-functional receptor expression

Proper interpretation of subcellular localization can provide insights into NR3C2 activity and function in different physiological and pathological contexts.