NR2C2 Antibody

What is NR2C2 and why is it important in research?

NR2C2, also known as TAK1, TR4, or TR2R1, belongs to the nuclear hormone receptor family and NR2 subfamily. It functions as an orphan nuclear receptor that regulates gene expression during the late phase of spermatogenesis. Recent research has revealed its significant role in inflammation, particularly in macrophages during bacterial infections. NR2C2 has been identified as a potential regulatory factor in testicular inflammatory injury by activating the NF-κB pathway, which promotes the expression of inflammatory cytokines such as IL-1β and IL-6 . The importance of studying NR2C2 stems from its involvement in multiple biological processes including reproduction, inflammation, and potentially fertility issues related to bacterial infections. Understanding NR2C2's functions can provide insights into the molecular mechanisms of inflammation-related male infertility and identify potential therapeutic targets for treatment approaches.

Which applications are NR2C2 antibodies validated for?

NR2C2 antibodies from various suppliers are validated for different applications based on rigorous testing. The most common applications include:

When selecting an NR2C2 antibody for your research, it's essential to check if it has been validated for your specific application . Cross-reactivity with your species of interest is also an important consideration, with available antibodies showing reactivity to human, mouse, rat, and monkey samples depending on the manufacturer .

What is the expected molecular weight of NR2C2 in Western blot applications?

When performing Western blot analysis with NR2C2 antibodies, researchers should be aware of the expected molecular weight to correctly identify the protein:

The discrepancy between calculated and observed molecular weights (65 kDa vs. 67-70 kDa) is not uncommon in protein analysis and may be due to post-translational modifications, protein folding characteristics, or the specific gel system used . When analyzing your Western blot results, look for bands in the 65-70 kDa range to identify NR2C2. Positive controls such as HeLa, Jurkat, or PC-3 cells have been confirmed to express NR2C2 and can be used to validate antibody specificity and performance .

What are the optimal protocols for using NR2C2 antibodies in Western blotting?

For optimal Western blotting results with NR2C2 antibodies, follow these methodological guidelines:

• Sample preparation:

  • Extract proteins using RIPA buffer supplemented with protease inhibitors

  • Determine protein concentration using Bradford or BCA assay

  • Load 20-40 μg of total protein per lane

• Gel electrophoresis:

  • Use 8-10% SDS-PAGE gels as NR2C2 has a molecular weight of 65-70 kDa

  • Include positive control samples (HeLa, Jurkat, or PC-3 cells have been confirmed to express NR2C2)

• Transfer and blocking:

  • Transfer to PVDF or nitrocellulose membrane at 100V for 60-90 minutes

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

• Antibody incubation:

  • Primary antibody dilution:

    • 1:500-1:3000 for Proteintech antibody (20981-1-AP)

    • 1:1000 for Cell Signaling Technology antibody (#68191)

  • Incubate overnight at 4°C

  • Wash 3-5 times with TBST, 5 minutes each

  • Secondary antibody: Anti-rabbit HRP-conjugated, diluted 1:5000-1:10000

  • Incubate for 1 hour at room temperature

• Detection:

  • Use ECL reagent for detection

  • Expected band: 65-70 kDa

These protocols should be optimized for your specific experimental conditions and sample types. Always include appropriate positive controls and consider running a gradient of antibody dilutions to determine the optimal concentration for your system.

How should NR2C2 antibodies be used for immunofluorescence studies?

Immunofluorescence (IF) staining with NR2C2 antibodies requires careful attention to fixation, permeabilization, and antibody concentrations. Based on published protocols:

• Sample preparation:

  • For tissue sections: Use 4 μm thick frozen sections embedded in OCT

  • For cultured cells: Grow cells on coverslips and fix with 4% paraformaldehyde for 15 minutes

• Blocking and permeabilization:

  • Wash sections/cells in PBS

  • Block for 1 hour at room temperature with 5% fetal bovine serum (FBS) and 0.3% Triton-X-100 in PBS

• Antibody incubation:

  • Primary antibody: Anti-NR2C2 (e.g., Bioss, #bs-4636R) at 1:200 dilution

  • Incubate overnight at 4°C

  • Wash 3 times with PBS, 5 minutes each

  • Secondary antibody: CoraLite488-conjugated AffiniPure goat anti-rabbit IgG (H+L) at 1:200 dilution

  • Incubate for 1 hour at room temperature

• Nuclear staining and mounting:

  • Wash 3 times with PBS, 5 minutes each

  • Counterstain nuclei with DAPI

  • Mount slides with anti-fade mounting medium

• Imaging:

  • Capture images using confocal microscopy

  • For co-localization studies, use appropriate markers such as F4/80 (macrophage marker) or DDX4 (germ cell marker)

This protocol has been successfully used to examine the expression and localization of NR2C2 in testicular tissue, particularly its expression in macrophages during inflammation studies.

What controls should be included when using NR2C2 antibodies?

Proper controls are essential for validating antibody specificity and ensuring reliable results:

• Positive controls:

  • Cell lines known to express NR2C2 (HeLa, Jurkat, PC-3 cells)

  • Tissues with known expression (testicular tissue)

• Negative controls:

  • Primary antibody omission control

  • Isotype control (normal rabbit IgG)

  • Samples with NR2C2 knockdown (siRNA or shRNA)

  • Tissue sections from NR2C2 knockout animals (if available)

• Blocking peptide control:

  • Pre-incubate the antibody with its specific immunizing peptide

  • This should abolish specific staining

• Cross-reactivity control:

  • Test the antibody on samples from different species to confirm cross-reactivity claims

  • Verify reactivity with human, mouse, rat, or monkey samples as applicable

• Loading controls for Western blotting:

  • Use housekeeping proteins such as β-actin, GAPDH, or tubulin

Including these controls helps validate antibody specificity and ensures that your observations are due to specific NR2C2 detection rather than non-specific binding or artifacts, which is particularly important when studying nuclear proteins that may have related family members with similar sequences.

How can I effectively knockdown NR2C2 to study its function in macrophages?

RNA interference techniques have been successfully used to knock down NR2C2 expression in macrophages to study its function. Based on published research:

• siRNA transfection:

  • Design or purchase validated siRNAs targeting NR2C2 mRNA

  • For RAW264.7 macrophages, transfection using commercial reagents such as Lipofectamine has been effective

  • Use 50-100 nM siRNA concentration

  • Include a non-targeting siRNA control

• Knockdown verification:

  • Confirm knockdown efficiency by Western blot using anti-NR2C2 antibody

  • Quantify protein reduction (aim for >70% reduction)

  • Also verify at the mRNA level using RT-qPCR

• Functional assays post-knockdown:

  • For inflammation studies, stimulate cells with LPS (typically 100 ng/ml) for 6-24 hours

  • Measure inflammatory cytokine production (IL-1β, IL-6) by ELISA or qPCR

  • Assess NF-κB pathway activation by Western blot (phospho-p65, IκBα degradation)

  • Collect conditioned media to study paracrine effects on other cell types

• Rescue experiments:

  • To confirm specificity, perform rescue experiments by re-expressing siRNA-resistant NR2C2

This approach has revealed that NR2C2 knockdown in macrophages downregulates the expression of inflammatory factors such as IL-1β and IL-6, and alleviates the inhibitory effect of inflammatory supernatant secreted by macrophages on the proliferation of spermatogonia .

How can I investigate the interaction between NR2C2 and the NF-κB pathway?

To study the molecular mechanisms of how NR2C2 activates the NF-κB pathway:

• Promoter binding analysis:

  • Use bioinformatics tools like EPD and JASPAR to predict NR2C2-binding sites (DR elements) in the promoters of NF-κB pathway genes

  • Perform dual luciferase reporter assays using the following approach:

    • Clone the promoter regions containing DR elements into pGL3 basic reporter plasmids

    • Co-transfect with NR2C2 expression vector (e.g., pcDNA3.1-FLAG-TR4)

    • Measure luciferase activity to quantify transcriptional activation

• Chromatin immunoprecipitation (ChIP):

  • Use anti-NR2C2 antibody to immunoprecipitate chromatin

  • Perform qPCR with primers spanning predicted binding sites

  • Include IgG control for background normalization

• Co-immunoprecipitation:

  • Investigate protein-protein interactions between NR2C2 and NF-κB subunits

  • Use anti-NR2C2 antibody for immunoprecipitation

  • Detect co-precipitated NF-κB proteins by Western blot

• Signaling pathway analysis:

  • Monitor phosphorylation status of key NF-κB pathway proteins

  • Assess nuclear translocation of p65 by subcellular fractionation or immunofluorescence

  • Use NF-κB pathway inhibitors to confirm specificity

Research has shown that NR2C2 activates NF-κB signaling by binding with DR elements in the promotor of the Nfκb gene, providing a molecular mechanism for its proinflammatory role in macrophages . This was demonstrated using dual luciferase reporter assays with the Nfκb1 promotor (−872 to −11) containing five NR2C2-binding sites and the Rela promotor (−753 to 244) containing four DR elements .

How can I perform co-localization studies of NR2C2 with cell-type specific markers?

Co-localization studies are valuable for determining which cell types express NR2C2 and its subcellular localization. For example, researchers have successfully co-localized NR2C2 with macrophage and germ cell markers in testicular tissue:

• Multi-color immunofluorescence protocol:

  • Prepare tissue sections or cells as described in the immunofluorescence protocol

  • Use a combination of primary antibodies from different host species:

    • Rabbit anti-NR2C2 (1:200, Bioss, #bs-4636R)

    • Mouse anti-F4/80 (1:50, macrophage marker)

    • Mouse anti-DDX4 (1:50, germ cell marker)

  • Incubate with species-specific secondary antibodies with different fluorophores:

    • CoraLite488-conjugated anti-rabbit IgG

    • CoraLite594-conjugated anti-mouse IgG

  • Counterstain nuclei with DAPI

  • Image using confocal microscopy with appropriate channel settings

• Analysis of co-localization:

  • Use specialized software (ImageJ with Coloc2 plugin, CellProfiler, etc.)

  • Calculate Pearson's or Mander's coefficients to quantify co-localization

  • Perform z-stack imaging to confirm co-localization in three dimensions

• Controls for co-localization:

  • Single-stained controls to establish proper channel settings

  • Non-expressing regions as negative controls

  • Fluorophore-swapped controls to rule out bleed-through artifacts

This approach has revealed that NR2C2 is upregulated specifically in testicular macrophages in LPS-induced mouse orchitis models, providing important insights into its cell-type specific function during inflammation .

How can I investigate the functional consequences of NR2C2 activity in macrophages?

To study the functional impact of NR2C2 in macrophages, particularly its role in inflammation and effects on neighboring cells:

• Conditioned media experiments:

  • Transfect macrophages with NR2C2 siRNA or control siRNA

  • Stimulate with LPS to induce inflammation

  • Collect conditioned media

  • Apply conditioned media to target cells (e.g., spermatogonia GC-1 SPG cells)

  • Assess proliferation using:

    • Cell counting kit-8 (CCK-8) analysis

    • 5-Ethynyl-2′-deoxyuridine (EdU) staining for DNA synthesis

  • Measure other functional parameters as appropriate for your research question

• Cytokine profiling:

  • Use multiplex ELISA or cytokine arrays to profile secreted factors

  • Correlate cytokine levels with NR2C2 expression

  • Neutralize specific cytokines to determine their contribution to observed effects

• Migration and invasion assays:

  • Study the impact of NR2C2-regulated inflammatory environment on cell migration

  • Use transwell assays with conditioned media from NR2C2-modified macrophages

• In vivo models:

  • Use macrophage-specific NR2C2 knockout or overexpression models

  • Induce inflammation (e.g., LPS injection)

  • Analyze tissue-specific effects and systemic responses

  • Assess long-term consequences on tissue function and recovery

Research has shown that knockdown of NR2C2 in macrophages alleviates the inhibitory effect of inflammatory supernatant on the proliferation of spermatogonia, suggesting that NR2C2-regulated inflammation in macrophages can impact the function of neighboring cells in the testicular microenvironment .

How do I interpret discrepancies in NR2C2 molecular weight across different studies?

Researchers often encounter variations in the observed molecular weight of NR2C2 in Western blot analyses. These discrepancies may be due to:

• Post-translational modifications:

  • Phosphorylation, SUMOylation, or ubiquitination can increase apparent molecular weight

  • Different cell types or conditions may exhibit different modification patterns

• Gel system variations:

  • Different percentage gels, buffer systems, or commercial pre-cast gels can affect migration

  • Gradient gels versus fixed percentage gels may show different apparent molecular weights

• Sample preparation:

  • Denaturation conditions (reducing agents, heating time)

  • Protein extraction methods may preserve or disrupt certain modifications

Based on available data:

• Calculated molecular weight: 65 kDa

• Observed molecular weight: 67-70 kDa

This discrepancy of 2-5 kDa is within the normal range of variation for nuclear receptors and likely reflects post-translational modifications or structural features that affect electrophoretic mobility. When troubleshooting molecular weight discrepancies, run positive control samples alongside your experimental samples and include samples with known NR2C2 overexpression if possible.

What are common issues in NR2C2 antibody-based experiments and how can they be resolved?

When working with NR2C2 antibodies, researchers may encounter several common issues:

• High background in Western blots:

  • Issue: Non-specific bands or smearing

  • Solution:

    • Increase blocking time or concentration (try 5% BSA instead of milk)

    • Reduce primary antibody concentration

    • Increase washing steps (number and duration)

    • Use fresh transfer buffer and blocking reagents

    • Try different membrane types (PVDF vs. nitrocellulose)

• Weak or no signal:

  • Issue: Inability to detect NR2C2

  • Solution:

    • Confirm NR2C2 expression in your sample using RT-qPCR

    • Increase protein loading (up to 50-60 μg per lane)

    • Reduce antibody dilution (use more concentrated antibody)

    • Use enhanced detection methods (high-sensitivity ECL)

    • Try a different antibody targeting a different epitope

• Multiple bands in immunoblotting:

  • Issue: Several bands of different sizes

  • Solution:

    • Verify with positive controls like HeLa, Jurkat, or PC-3 cells

    • Use freshly prepared samples to minimize degradation

    • Include protease inhibitors in lysis buffer

    • The band at 65-70 kDa is the full-length NR2C2; other bands may be isoforms, degradation products, or non-specific binding

• Poor immunofluorescence staining:

  • Issue: Weak signal or high background

  • Solution:

    • Optimize fixation and permeabilization conditions (5% FBS and 0.3% Triton-X-100)

    • Try antigen retrieval methods for tissue sections

    • Increase antibody concentration or incubation time

    • Use signal amplification systems if necessary

    • Include appropriate controls to distinguish specific from non-specific staining

Addressing these issues systematically will improve the reliability and reproducibility of your NR2C2 antibody-based experiments.

How is NR2C2 being studied in the context of inflammation and immunity?

Recent research has revealed important roles for NR2C2 in inflammation and immune responses:

• Macrophage activation and polarization:

  • NR2C2 is upregulated in macrophages upon LPS stimulation

  • It promotes proinflammatory responses through NF-κB activation

  • Research has shown it regulates the expression of inflammatory factors such as IL-1β and IL-6

• Cytokine regulation:

  • The molecular mechanisms by which NR2C2 regulates cytokine expression involve binding to DR elements in the promotor regions of key inflammatory genes

  • NR2C2 activates NF-κB signaling by binding with DR elements in the promotor of the Nfκb gene

• Tissue-specific inflammatory responses:

  • Initial studies have focused on testicular inflammation and orchitis

  • NR2C2 has been found to be highly expressed in the testes and upregulated in testicular macrophages in LPS-induced mouse orchitis models

  • This suggests a specific role in reproductive inflammation

• Impact on neighboring cells:

  • The inflammatory environment created by NR2C2-expressing macrophages affects the function of neighboring cells

  • Knockdown of NR2C2 in macrophages alleviates the inhibitory effect of inflammatory supernatant on the proliferation of spermatogonia

  • This reveals a paracrine mechanism by which NR2C2-regulated inflammation impacts tissue function

As a nuclear receptor, NR2C2 represents an intriguing target for modulating inflammatory responses, potentially offering more selective approaches than broad immunosuppressive therapies, particularly in the context of reproductive inflammation and infection-induced male infertility.

What are the future directions for NR2C2 antibody applications in research?

As research on NR2C2 continues to evolve, several promising directions for antibody applications are emerging:

• Development of highly specific monoclonal antibodies:

  • Current commercial antibodies are primarily polyclonal

  • Next-generation monoclonal antibodies with higher specificity would improve detection in complex tissues

  • Epitope-mapped antibodies targeting different domains of NR2C2 could help distinguish different functional states of the protein

• Advanced imaging applications:

  • Super-resolution microscopy to study NR2C2 nuclear distribution patterns

  • Live-cell imaging using fluorescently tagged anti-NR2C2 antibody fragments

  • Multiplex immunofluorescence combining NR2C2 with other nuclear factors and signaling molecules

• Therapeutic and diagnostic development:

  • Using NR2C2 antibodies to develop assays for monitoring inflammation in reproductive disorders

  • Potential development of blocking antibodies that could modulate NR2C2 function in inflammatory conditions

  • Companion diagnostics for future NR2C2-targeting therapeutics

• Single-cell applications:

  • Integration with single-cell proteomics to study cell-to-cell variation in NR2C2 expression

  • Application in spatial proteomics to understand NR2C2 distribution in complex tissues

  • Development of CyTOF/mass cytometry compatible antibodies for high-parameter analysis

These advances in antibody technology and applications will further our understanding of NR2C2's role in normal physiology and disease states, potentially leading to new diagnostic and therapeutic approaches for inflammation-related disorders.