NR2C1 Antibody

What is NR2C1 and what are its primary structural domains?

NR2C1 is a nuclear hormone receptor that functions as a transcription factor. It belongs to a large family of ligand-inducible transcription factors that regulate gene expression by binding to specific DNA sequences within promoters of target genes. The protein is characterized by three primary structural domains: a highly conserved DNA binding domain (DBD), a variable hinge region, and a carboxy-terminal ligand binding domain (LBD) . These structural elements are typical for all members of the steroid/thyroid hormone receptor superfamily. The protein has multiple alternatively spliced transcript variants, though the full-length nature of some variants remains undetermined .

What cellular functions does NR2C1 regulate?

NR2C1 functions primarily as a repressor for a broad range of genes, though it can also activate specific gene expressions. It binds to hormone response elements (HREs) consisting of two 5'-AGGTCA-3' half site direct repeat consensus sequences . It plays key roles in:

• Stem cell proliferation and differentiation

• Autoregulatory negative feedback mechanisms through binding to the IR7 element in its own promoter

• Activation of OCT4 gene expression (important in pluripotency)

• Retinoic acid-regulated preadipocyte proliferation

• Repression of embryonic and fetal globin transcription (when paired with NR2C2 in the DRED complex)

How should I store and handle NR2C1 antibodies for optimal performance?

NR2C1 antibodies should be stored at -20°C and freeze/thaw cycles should be avoided to maintain antibody integrity and performance . Before opening the vial, it's recommended to centrifuge to ensure complete recovery of the contents. The antibody is typically supplied in PBS buffer with 0.02% sodium azide and 50% glycerol at pH 7.3 . When working with the antibody, proper laboratory practices should be observed including using appropriate personal protective equipment due to the presence of sodium azide, which is a toxic preservative.

What are the recommended applications and dilutions for NR2C1 antibodies?

Based on validated research protocols, NR2C1 antibodies are primarily used in:

• Western Blot (WB) analysis: Recommended dilution range of 1:500-1:2000, with 1:1000 being commonly used for cell line extracts

• Immunofluorescence (IF): Recommended dilution range of 1:50-1:200

The observed molecular weight in Western blot analyses is approximately 75kDa, which differs from the calculated molecular weights of 51kDa/53kDa/67kDa, possibly due to post-translational modifications .

How can I optimize transfection protocols when studying NR2C1 function?

When studying NR2C1 function through gene manipulation, transfection protocols similar to those used for related nuclear receptors have proven effective. For plasmid-based transfections:

• Incubate plasmids with Lipofectamine™ 3000 reagents and Opti-MEM™ for approximately 20 minutes at room temperature

• Use appropriate plasmid concentrations based on culture dish size (e.g., 6 μg for 35 mm dishes and 4 μg for 15 mm dishes)

• For co-transfection experiments, combine plasmids before adding transfection reagents

• Harvest cells approximately 36 hours post-transfection for subsequent experiments

This approach has been successfully employed in studies examining the functional relationship between nuclear receptors and their regulatory targets.

What controls should I include when validating NR2C1 antibody specificity?

To ensure antibody specificity and experimental validity:

• Positive controls: Include cell lines known to express NR2C1, such as MCF7 cells, which have been validated for NR2C1 expression

• Negative controls: Either use cell lines with NR2C1 knockdown or tissues known not to express significant levels of NR2C1

• Antibody controls: Include an isotype control (rabbit IgG) to assess non-specific binding

• Knockout validation: When possible, use CRISPR/Cas9 engineered cell lines with NR2C1 knockout to confirm antibody specificity

• Peptide competition: Pre-incubate the antibody with the immunizing peptide to confirm binding specificity

Additionally, comparing results from antibodies raised against different epitopes of NR2C1 can further validate specificity.

How does NR2C1 contribute to stem cell biology and pluripotency regulation?

NR2C1 has emerged as a significant modulator of pluripotentiality, particularly during hominid evolution. Experimental studies have identified NR2C1 as a regulator of key pluripotency factors:

• NR2C1 functions as an activator of OCT4 gene expression, a master regulator of pluripotency

• It influences the transcriptional modulation of pluripotency factors including OCT4 and NANOG

• It affects the average size of embryological stem cell colonies, serving as a proxy for the self-renewal capacity of pluripotent cells

• Evolutionary analyses have revealed adaptive changes in NR2C1 that coincide with alterations in pluripotentiality regulation within primates

These findings suggest that NR2C1 plays a crucial role in maintaining stem cell characteristics and may be involved in the evolutionary divergence of pluripotency regulation mechanisms.

What is the relationship between NR2C1 and cancer biology, particularly in breast cancer?

NR2C1 and related nuclear receptors have been implicated in cancer biology through several mechanisms:

• NR2C1 may interact with the NR2E3/NR2C2 signaling pathway in estrogen receptor-positive (ER+) breast cancer cells

• This signaling pathway appears to modulate stem-like properties of cancer cells, including the CD44+CD24-/low cell population ratio and migratory activity

• NR2C1/NR2C2 interactions influence cancer cell phenotypes in ways that may affect tumor progression and therapeutic responses

Research suggests that NR2C1 and related nuclear receptors might serve as potential therapeutic targets in ER+ breast cancer, particularly through their effects on cancer stem cell-like properties .

How can I analyze NR2C1 expression patterns across different tissues and disease states?

For comprehensive analysis of NR2C1 expression:

• Transcriptomic analysis: Utilize databases like NIH/NCBI UniGene to analyze expression profiles across different tissues. Normalize transcripts per million of genes of interest to housekeeping genes like β-actin to calculate arbitrary units of gene expression

• Comparative expression analysis: Implement the following methodological approach:

  • Compare expression levels across 21+ human and 17+ mouse tissues including heart and vasculature

  • Normalize to consistent housekeeping genes across all samples

  • Apply statistical analysis to identify significant differences between tissue types

• Disease state analysis: When comparing normal versus pathological conditions:

  • Consider that most nuclear receptors, potentially including NR2C1, have less tendency to be upregulated than downregulated in cancers, autoimmune and metabolic diseases

  • Analyze regulation by inflammation pathways and mitochondrial energy enzymes

  • Assess the impact of the innate immune sensor inflammasome/caspase-1 pathway, which has been shown to regulate the expression of most nuclear receptors

How has NR2C1 evolved among primates and what functional implications does this have?

Research on NR2C1 evolution has revealed significant insights:

• Fixed-effect (FE) codon models characterize average rates and patterns of primate NR evolution with respect to its primary structural domains

• Branch-site codon models (developed to detect episodes of positive selection) and clade-site models (designed to detect changes in selection pressure distribution) have identified hominid-specific alterations in natural selection intensity affecting NR2C1

• Experimental validation has confirmed that adaptive evolution of gene regulation has impacted several aspects of pluripotentiality within primates through NR2C1 functional changes

These evolutionary changes suggest that NR2C1 has undergone adaptive modifications that correlate with functional divergence in gene regulation, particularly affecting pluripotency networks in hominid evolution.

What are the transcriptional signatures associated with NR2C1 dysregulation?

While specific NR2C1 transcriptional signatures require further characterization, broader nuclear receptor dysregulation patterns provide insights:

• Nuclear receptors show differential expression across tissues, potentially regulated by:

  • Oxygen sensors

  • Angiogenesis pathways

  • Stem cell master regulators

  • Inflammasomes

  • Tissue hypo-/hypermethylation indexes

• Nuclear receptor sequence mutations associate with increased risks for:

  • Cancer development

  • Metabolic diseases

  • Cardiovascular diseases

  • Autoimmune diseases

• Most nuclear receptors, potentially including NR2C1, demonstrate less tendency to be upregulated than downregulated in pathological conditions like cancers, autoimmune and metabolic diseases

These patterns suggest that NR2C1 dysregulation may contribute to distinct transcriptional signatures that could serve as potential biomarkers or therapeutic targets.

Why might there be discrepancies between observed and calculated molecular weights for NR2C1?

The observed molecular weight of NR2C1 in Western blot analyses (approximately 75kDa) differs significantly from the calculated molecular weights (51kDa/53kDa/67kDa) . These discrepancies could result from:

• Post-translational modifications: Phosphorylation, glycosylation, SUMOylation, or ubiquitination can significantly alter protein migration

• Alternative splicing: Multiple transcript variants of NR2C1 have been described, though the full-length nature of some variants remains undetermined

• Protein-protein interactions: Strong interactions resistant to SDS denaturation

• Technical factors: Choice of gel percentage, running buffer composition, and marker calibration

• Structural characteristics: Highly charged regions or hydrophobic domains can affect SDS binding and protein migration

To address these discrepancies, researchers should consider employing mass spectrometry to confirm protein identity and molecular weight, and use multiple antibodies targeting different epitopes to validate results.

How can I optimize co-immunoprecipitation protocols for studying NR2C1 interactions with other nuclear receptors?

Effective co-immunoprecipitation (Co-IP) of NR2C1 with interacting partners requires:

• Cell lysis optimization:

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

  • Include protease and phosphatase inhibitors to prevent protein degradation

  • Maintain cold temperatures throughout the procedure

• Pre-clearing step:

  • Incubate lysates with protein A/G beads to reduce non-specific binding

  • Remove beads by centrifugation before adding the specific antibody

• Antibody selection and incubation:

  • Use 2-5μg of anti-NR2C1 antibody per 500μg of protein lysate

  • Allow overnight incubation at 4°C with gentle rotation

• Washing and elution:

  • Perform at least 4-5 washes with decreasing salt concentrations

  • Elute using either SDS sample buffer (denaturing) or specific peptides (native)

• Detection strategies:

  • Analyze by Western blot using antibodies against suspected interaction partners

  • Consider mass spectrometry for unbiased identification of interacting proteins

When specifically studying interactions with other nuclear receptors like NR2C2 or NR2E3, ensure cross-reactivity between antibodies is minimized.

How can NR2C1 research contribute to our understanding of inflammatory and metabolic diseases?

Recent findings suggest nuclear receptors, including NR2C1, may function as anti-inflammatory homeostasis-associated molecular pattern receptors (HAMPRs) . This paradigm offers several research directions:

• Inflammatory regulation: Investigate how NR2C1 interacts with the inflammasome/caspase-1 pathway, which has been shown to regulate the expression of most nuclear receptors

• Metabolic disease: Examine NR2C1's role in:

  • Adipocyte differentiation and function

  • Energy metabolism regulation

  • Insulin sensitivity and glucose homeostasis

• Therapeutic targeting: Develop approaches that modulate NR2C1 activity to address inflammatory and metabolic disorders

• Biomarker development: Assess whether NR2C1 expression patterns or post-translational modifications could serve as biomarkers for disease progression or treatment response

These research directions could significantly advance our understanding of how nuclear receptors like NR2C1 integrate metabolic and inflammatory signaling networks.

What role might NR2C1 play in epigenetic regulation and how can this be studied experimentally?

NR2C1's potential role in epigenetic regulation presents an exciting research frontier:

• Chromatin immunoprecipitation sequencing (ChIP-seq):

  • Map genome-wide binding sites of NR2C1

  • Correlate binding with histone modifications and chromatin accessibility

  • Identify target genes and regulatory networks

• ATAC-seq integration:

  • Combine NR2C1 ChIP-seq with ATAC-seq to assess how NR2C1 influences chromatin accessibility

  • Determine whether NR2C1 functions primarily at open or closed chromatin regions

• Histone modification analysis:

  • Examine how NR2C1 binding correlates with specific histone marks (H3K4me3, H3K27ac, H3K27me3)

  • Assess whether NR2C1 recruits histone-modifying enzymes to target loci

• DNA methylation studies:

  • Investigate potential interactions between NR2C1 and DNA methylation machinery

  • Analyze whether NR2C1 binding is influenced by DNA methylation status

These approaches could reveal how NR2C1 contributes to the epigenetic regulation of gene expression in development, differentiation, and disease.