NCOR2 Antibody

What is NCOR2 and why is it significant in research?

NCOR2, also known as SMRT (Silencing Mediator for Retinoid and Thyroid hormone receptors), TRAC-2, and CTG26, is a 265-275 kDa transcriptional co-repressor protein. Human NCOR2 is 2525 amino acids in length and contains multiple functional domains, including two DNA-binding SANT domains (at amino acids 427-478 and 610-661) and coiled-coil regions (at amino acids 174-215 and 522-561) .

NCOR2 is significant in research because it functions as a transcriptional repressor by forming complexes with histone deacetylases (HDACs), particularly HDAC3, as well as with TBL1 and GPS2. This complex interacts with nuclear hormone receptors to regulate gene expression . Recent studies have revealed NCOR2's critical roles in:

• Cancer progression, particularly in prostate cancer where reduced NCOR2 expression accelerates disease recurrence following androgen deprivation therapy (ADT)

• Drug resistance development in multiple myeloma, where low NCOR2 levels drive multidrug resistance

• Epigenetic regulation through DNA methylation and histone modification

These functions make NCOR2 an important research target for understanding disease mechanisms and developing potential therapeutic strategies.

How do NCOR2 antibodies contribute to understanding NCOR2's role in disease?

NCOR2 antibodies are essential tools for investigating NCOR2's function in various pathological conditions. In prostate cancer research, NCOR2 antibodies have revealed that reduced NCOR2 expression significantly associates with shorter disease-free survival in patients receiving androgen deprivation therapy (ADT) . Methodologically, researchers can apply NCOR2 antibodies in multiple techniques:

• Immunohistochemistry (IHC) to assess NCOR2 expression levels in clinical samples, as demonstrated in the 707-patient tissue microarray study of radical prostatectomy samples

• Immunofluorescence (IF) to examine subcellular localization of NCOR2

• Western blotting to quantify NCOR2 protein levels

• Chromatin immunoprecipitation (ChIP) to map NCOR2 binding sites genome-wide

For example, in breast cancer tissue studies, NCOR2 antibodies have demonstrated nuclear localization in epithelial cells through immunohistochemical staining . The application of validated NCOR2 antibodies has directly contributed to findings that NCOR2 reduction accelerates disease recurrence following ADT from a median of 232 days to 180 days in xenograft models .

What are the key considerations when selecting an NCOR2 antibody?

When selecting an NCOR2 antibody for research, several technical factors must be considered:

Specificity: Choose antibodies with validated specificity for NCOR2 with minimal cross-reactivity. For example, some commercial NCOR2 antibodies show less than 1% cross-reactivity with the structurally similar NCOR1 in direct ELISAs . This is crucial because NCOR1 and NCOR2 share functional similarities but have distinct biological roles.

Target region: Select antibodies targeting specific domains based on your research question. Available options include:

• Antibodies targeting amino acids 511-560

• Antibodies targeting amino acids 2268-2449

• Antibodies targeting the N-terminus or C-terminus

Validated applications: Ensure the antibody is validated for your specific application:

• ELISA detection of NCOR2

• IHC in paraffin-embedded tissues

• Immunofluorescence studies

• Western blotting

Host species: Consider the host species (most commonly rabbit or mouse) to avoid cross-reactivity in multi-labeling experiments .

Clonality: Polyclonal antibodies offer broader epitope recognition, while monoclonal antibodies provide higher specificity for a single epitope .

Proper antibody selection directly impacts experimental outcomes, particularly in detecting NCOR2 in different cellular compartments and protein complexes.

What protocols are recommended for NCOR2 detection in tissue samples?

For optimal NCOR2 detection in tissue samples, the following methodological approaches are recommended:

Immunohistochemistry (IHC) protocol:

• Fixation: Use formalin fixation for paraffin-embedded sections of tissue samples

• Antigen retrieval: Heat-induced epitope retrieval in citrate buffer (pH 6.0)

• Antibody concentration: 15 μg/mL for optimal staining, incubated overnight at 4°C

• Detection system: HRP-DAB system for visualization

• Counterstaining: Hematoxylin for nuclear contrast

• Result interpretation: NCOR2 typically shows nuclear localization in epithelial cells

Immunofluorescence (IF) protocol:

• Cell fixation: 4% paraformaldehyde for 10-15 minutes

• Permeabilization: 0.1% Triton X-100 for 5-10 minutes

• Blocking: 5% normal serum (matching secondary antibody host) for 1 hour

• Primary antibody: Anti-NCOR2 antibody (e.g., targeting amino acids 511-560) at manufacturer-recommended dilution, incubated overnight at 4°C

• Secondary antibody: Fluorophore-conjugated secondary antibody for 1-2 hours at room temperature

• Counterstaining: DAPI for nuclear visualization

• Imaging: Confocal microscopy for co-localization studies

When analyzing clinical samples, researchers should consider using a quantitative scoring method such as H-score for NCOR2 expression, which combines staining intensity and percentage of positive cells, as used in the 707-patient tissue microarray study .

How can researchers optimize ChIP protocols for studying NCOR2 genomic interactions?

Chromatin immunoprecipitation (ChIP) is crucial for understanding NCOR2's genomic interactions, particularly its role in transcriptional regulation. An optimized ChIP protocol includes:

Fixation and chromatin preparation:

• Cross-link cells with 1% formaldehyde for 10 minutes at room temperature

• Quench with 125 mM glycine for 5 minutes

• Lyse cells and isolate nuclei

• Sonicate chromatin to 200-500 bp fragments (optimize sonication time for each cell type)

• Pre-clear chromatin with protein A/G beads

Immunoprecipitation:

• Incubate chromatin with 2-5 μg of validated NCOR2 antibody overnight at 4°C

• Add protein A/G beads and incubate for 2-4 hours

• Wash beads stringently (low-salt, high-salt, LiCl, and TE washes)

• Elute protein-DNA complexes and reverse cross-links

• Purify DNA for downstream analysis

Data analysis considerations:

• Include appropriate controls (input DNA, IgG control)

• For ChIP-seq, use spike-in controls for normalization

• Analyze NCOR2 binding in relation to histone modifications (H3K9me3) and DNA methylation patterns

• Focus analysis on enhancer regions, where NCOR2 binding most clearly associates with transcriptional regulation

Research has shown that NCOR2 genomic binding is most pronounced in androgen-independent cells (e.g., LNCaP-C4-2) and frequently associates with forkhead box (FOX) transcription factor FOXA1 binding sites . When designing ChIP-seq experiments, researchers should include analyses of these known co-factor binding patterns.

What strategies can overcome challenges in detecting low NCOR2 expression levels?

Detecting low levels of NCOR2 expression can be challenging, particularly in clinical samples or when studying drug-resistant cells. The following strategies can enhance detection sensitivity:

Signal amplification methods:

• Tyramide signal amplification (TSA) for IHC and IF, which can increase sensitivity 10-100 fold

• Use of high-sensitivity ECL substrates for Western blotting

• Proximity ligation assay (PLA) for detecting NCOR2 interactions with other proteins

Enrichment strategies:

• Nuclear fractionation to concentrate NCOR2 protein before detection

• Immunoprecipitation followed by Western blotting for low-abundance samples

• Nested PCR approaches for mRNA detection

Quantification methods:

• Digital PCR for precise quantification of low NCOR2 mRNA levels

• Quantitative image analysis for IHC using appropriate software (e.g., ImageJ with IHC profiler plugin)

• Mass spectrometry-based approaches for absolute quantification

Technical considerations:

• Freshly prepared samples yield better results than archived samples

• Optimize antibody concentration through titration experiments

• Extend primary antibody incubation time (e.g., 48-72 hours at 4°C for IHC of difficult samples)

• Consider using antibodies targeting different NCOR2 epitopes to confirm results

These strategies are particularly important when studying NCOR2 downregulation in drug-resistant cancer cells, as observed in lenalidomide-resistant and pomalidomide-resistant multiple myeloma cell lines .

How should researchers interpret discrepancies between NCOR2 mRNA and protein levels?

Discrepancies between NCOR2 mRNA and protein levels are frequently observed in research and require careful interpretation:

Possible mechanisms explaining discrepancies:

• Post-transcriptional regulation: microRNAs targeting NCOR2 mRNA

• Post-translational modifications affecting protein stability

• Protein degradation pathways (ubiquitin-proteasome system)

• Alternative splicing generating different isoforms detected with varying efficiency

• Technical limitations in detection methods

Analytical approach:

• Validate findings using multiple techniques (qRT-PCR, Western blot, immunohistochemistry)

• Use antibodies targeting different epitopes to confirm protein results

• Assess temporal dynamics (mRNA changes often precede protein changes)

• Examine known regulators of NCOR2 stability

• Consider cell-type specific differences in post-transcriptional regulation

In multiple myeloma research, for example, NCOR2 downregulation has been observed at both mRNA and protein levels in drug-resistant cell lines, but the mechanisms driving this downregulation may include both transcriptional repression and enhanced protein degradation . Similarly, in prostate cancer, reduced NCOR2 expression significantly associates with shorter disease-free survival in patients receiving ADT, highlighting the importance of accurate protein level assessment in clinical samples .

What controls are essential when studying NCOR2 interactions with epigenetic regulators?

When investigating NCOR2 interactions with epigenetic regulators like histone deacetylases (HDACs), several controls are essential for proper data interpretation:

Positive controls:

• Known NCOR2-interacting proteins (e.g., HDAC3, TBL1, GPS2)

• Cell lines with confirmed NCOR2-HDAC interactions

• Recombinant proteins with established interaction profiles

Negative controls:

• IgG control for co-immunoprecipitation

• Cell lines with NCOR2 knockout or knockdown

• Proteins known not to interact with NCOR2

Validation controls:

• Reciprocal co-immunoprecipitation (IP with NCOR2 antibody and blot for HDAC3, then IP with HDAC3 antibody and blot for NCOR2)

• Proximity ligation assay to visualize interactions in situ

• FRET or BiFC for live-cell interaction analysis

• Mass spectrometry to identify interaction partners unbiasedly

Functional controls:

• HDAC activity assays in the presence/absence of NCOR2

• Chromatin immunoprecipitation to confirm co-localization at specific genomic loci

• Gene expression analysis following disruption of the interaction

Research has established that NCOR2 forms complexes with histone deacetylases (particularly HDAC3) and allosterically interacts with them to promote repressive histone marks such as H3K9me3 . These interactions are critical for NCOR2's role in recruiting CpG methylation machinery, making proper controls essential for understanding the functional consequences of these interactions.

How can researchers distinguish between different NCOR2 protein complexes?

NCOR2 functions within different protein complexes to regulate gene expression. Distinguishing between these complexes requires specialized experimental approaches:

Sequential immunoprecipitation approach:

• First IP with NCOR2 antibody

• Elute complexes under mild conditions

• Second IP with antibodies against suspected complex components

• Analyze resulting proteins by Western blot or mass spectrometry

Density gradient separation:

• Extract nuclear protein complexes

• Separate by size using sucrose or glycerol gradient ultracentrifugation

• Analyze fractions for NCOR2 and potential partners

• Compare elution profiles to identify distinct complexes

Crosslinking mass spectrometry (XL-MS):

• Crosslink protein complexes in living cells

• Purify NCOR2-containing complexes

• Analyze by mass spectrometry to identify crosslinked peptides

• Reconstruct complex architecture based on crosslink distances

Analytical considerations:

• Different NCOR2 complexes may include:

  • NCOR2-HDAC3-TBL1-GPS2 (classical corepressor complex)

  • NCOR2-KAISO complex (associated with DNA methylation)

  • NCOR2-SHARP-lncRNA complex

  • NCOR2-AR (androgen receptor) complex

  • NCOR2-NuRD complex (in multiple myeloma)

Research has shown that NCOR2 can function as both a co-repressor and co-activator depending on its complex partners. For instance, in prostate cancer, NCOR2 can actively enhance transcription by androgen receptor (AR) , while in multiple myeloma, NCOR2 interacts with the nucleosome remodeling and deacetylase (NuRD) complex and represses CD180 expression, ultimately affecting MYC expression .

How can NCOR2 antibodies be used to study drug resistance mechanisms?

NCOR2 antibodies are valuable tools for investigating drug resistance mechanisms, particularly in cancer:

Methodological approaches:

• Expression profiling in resistant vs. sensitive cells:

  • Immunohistochemistry to quantify NCOR2 levels in patient samples

  • Western blotting to track NCOR2 expression changes during resistance development

  • Flow cytometry for single-cell analysis of NCOR2 expression in heterogeneous populations

• Genomic studies:

  • ChIP-seq to map changes in NCOR2 binding sites in resistant cells

  • CUT&RUN for higher resolution mapping of genomic interactions

  • Integration with DNA methylation data to identify epigenetic changes

• Protein interaction studies:

  • Co-immunoprecipitation to identify altered protein interactions in resistant cells

  • Proximity ligation assay to visualize changes in NCOR2 complexes in situ

Research applications in multiple myeloma:
Studies have shown that NCOR2 downregulation is associated with multidrug resistance in multiple myeloma. NCOR2 knockout led to high MYC expression and conferred resistance to pomalidomide, BET, and HDAC inhibitors, independent of Cereblon (CRBN) . Lenalidomide-resistant and pomalidomide-resistant myeloma cell lines acquired exonic mutations in NCOR2, showing both NCOR2 downregulation and MYC upregulation .

Research applications in prostate cancer:
Reduced NCOR2 expression significantly associates with shorter disease-free survival in prostate cancer patients receiving adjuvant ADT . In CWR22 xenograft models, knockdown of NCOR2 reduced time to recurrence following ADT from 232 days to 180 days, suggesting a role in therapy resistance .

Researchers can use NCOR2 antibodies to develop potential biomarkers for drug resistance and to identify novel therapeutic targets in treatment-resistant cancers.

What are the best approaches for studying NCOR2's role in epigenetic regulation?

NCOR2 plays a significant role in epigenetic regulation through various mechanisms. The following approaches are recommended for studying these functions:

Chromatin modification analysis:

• ChIP-seq for NCOR2 and histone modifications (particularly H3K9me3)

• CUT&Tag for higher sensitivity profiling of chromatin-associated factors

• ATAC-seq to identify changes in chromatin accessibility upon NCOR2 modulation

• Co-localization analysis with histone modifiers using sequential ChIP (ChIP-reChIP)

DNA methylation studies:

• Bisulfite sequencing to assess DNA methylation changes at NCOR2 binding sites

• Methylation-specific PCR for targeted analysis of specific loci

• Whole-genome bisulfite sequencing to identify global methylation patterns

• Integration of methylation data with NCOR2 ChIP-seq data

Functional genomics approaches:

• CRISPR interference or activation at NCOR2 binding sites

• HDAC inhibitor studies combined with NCOR2 modulation

• Gene expression analysis following NCOR2 knockout/knockdown

• Rescue experiments with NCOR2 mutants lacking specific interaction domains

Key findings to consider:
Research has shown that NCOR2 allosterically interacts with histone deacetylases to promote repressive histone marks such as H3K9me3, which then recruit CpG methylation machinery . Additionally, NCOR2 interacts with KAISO and with the long non-coding RNA SHARP to trigger DNA methylation . In prostate cancer studies, stably reduced NCOR2 expression induces global DNA hypermethylation patterns .

These methodologies enable researchers to dissect NCOR2's multifaceted roles in shaping the epigenome and regulating gene expression in normal and disease states.

How can multiplexed imaging techniques be applied to study NCOR2 in the tissue microenvironment?

Advanced multiplexed imaging techniques offer powerful approaches to study NCOR2 in complex tissue microenvironments:

Cyclic immunofluorescence (CycIF) approach:

• Stain tissue section with NCOR2 antibody and selected markers

• Image the section

• Chemically quench fluorophores

• Repeat with additional antibodies (up to 30-40 markers on the same section)

• Computational alignment and analysis of all markers

Mass cytometry imaging (MIBI/IMC) protocol:

• Label antibodies with isotopes rather than fluorophores

• Apply to tissue section

• Ablate tissue with ion beam and collect isotopes with mass spectrometer

• Generate high-dimensional spatial data of protein expression

• Analyze using computational spatial statistics

Spatial transcriptomics integration:

• Perform multiplexed protein imaging for NCOR2 and key interactors

• On adjacent sections, conduct spatial transcriptomics

• Computationally align protein and RNA data

• Correlate NCOR2 protein levels with local transcriptional profiles

Analytical considerations:

• Assess NCOR2 expression relative to cell type markers

• Quantify nuclear vs. cytoplasmic localization in different cell types

• Analyze co-expression with interaction partners (HDACs, nuclear receptors)

• Examine spatial relationships between NCOR2-expressing cells and other features in the tumor microenvironment

These approaches are particularly valuable for cancer studies, where NCOR2 expression has been associated with disease progression and therapy response. For example, in prostate cancer, NCOR2 expression varies based on clinical features including race, BMI, presurgical PSA, and Gleason sum . Multiplexed imaging could reveal how these clinical features correlate with NCOR2 expression in specific cell types within the tumor microenvironment.

What methodological approaches can identify novel NCOR2 functions in disease progression?

To identify novel NCOR2 functions in disease progression, researchers should consider these integrated methodological approaches:

Functional genomics screening:

• CRISPR-Cas9 screening of NCOR2-interacting partners

• Domain-specific CRISPR interference to disrupt specific NCOR2 functions

• Synthetic lethality screens in NCOR2-modulated backgrounds

• Pharmacological screening to identify compounds affecting NCOR2-dependent pathways

Systems biology approaches:

• Multi-omics integration (transcriptomics, proteomics, epigenomics)

• Network analysis to identify NCOR2-centered regulatory networks

• Machine learning to predict NCOR2-dependent disease outcomes

• Patient stratification based on NCOR2 expression patterns

Translational research models:

• Patient-derived xenografts with NCOR2 modulation

• Organoid models to study NCOR2 in 3D tissue architecture

• Genetically engineered mouse models with tissue-specific NCOR2 alterations

• Ex vivo patient sample manipulation and culture

Clinical correlation studies:

• Analyzing NCOR2 expression in patient cohorts with long-term follow-up

• Correlating NCOR2 levels with response to specific therapies

• Investigating NCOR2 mutations in treatment-resistant disease

Research has already identified several important NCOR2 functions in disease:

• In prostate cancer, reduced NCOR2 expression accelerates disease recurrence following androgen deprivation therapy and associates with gene expression patterns that include neuroendocrine features

• In multiple myeloma, NCOR2 knockout leads to high MYC expression and confers resistance to multiple drugs, while NCOR2 interacts with the NuRD complex and represses CD180 expression

These findings highlight the importance of integrated approaches to fully understand NCOR2's complex roles in disease progression and treatment response.