NSMCE3 Antibody

What is NSMCE3 and why is it significant in cellular biology?

NSMCE3 (NSE3 homolog, SMC5-SMC6 complex component) is a protein encoded by the NSMCE3 gene that forms an essential part of the structural maintenance of chromosomes (SMC) 5/6 complex. This complex plays crucial roles in DNA damage response and proper chromosome segregation during cell division.

Within the SMC5/6 complex, NSMCE3 interacts with NSMCE1 through its N-terminal winged-helix (WH) domain, WH/A, and with NSMCE4 via its C-terminal WH domain, WH/B. These interactions are essential for the formation of a tight subcomplex that bridges the large SMC5 and SMC6 subunits . The significance of NSMCE3 is highlighted by the fact that mutations disrupting these interactions can lead to destabilization of the entire SMC5/6 complex, resulting in chromosome rearrangements, micronuclei formation, sensitivity to replication stress, and defective homologous recombination .

What applications are NSMCE3 antibodies commonly used for?

NSMCE3 antibodies are utilized across multiple experimental applications:

When selecting an antibody, researchers should consider both the application needs and the specific epitope targeted by the antibody. Many commercial antibodies target the middle region of NSMCE3, which may affect detection depending on the experimental context .

How can I validate the specificity of an NSMCE3 antibody?

Validating antibody specificity is crucial for reliable results. For NSMCE3 antibodies, consider these methodological approaches:

• siRNA knockdown control: Use siRNA targeting NSMCE3 to reduce endogenous levels and confirm corresponding reduction in antibody signal. Published studies have shown that NSMCE3 antibodies detect endogenous levels that are reduced by siRNA .

• Overexpression control: Express recombinant tagged NSMCE3 (e.g., GFP or HA-tagged) and confirm detection with both the tag-specific antibody and the NSMCE3 antibody.

• Knockout cell lines: If available, use NSMCE3 knockout cell lines as negative controls, although complete knockout may be lethal given NSMCE3's essential functions.

• Peptide competition: Pre-incubate the antibody with the immunizing peptide to block specific binding sites.

• Cross-reactivity testing: Test reactivity against other SMC5/6 complex components to ensure specificity.

When reporting validation results, include all controls and document the specific batches and dilutions of antibodies used for reproducibility .

What are the optimal conditions for detecting NSMCE3 in Western blots?

For optimal Western blot detection of NSMCE3, consider the following protocol parameters:

Sample preparation:

• Whole-cell extracts should be obtained by sonication in strong lysis buffers such as UTB buffer (8 M urea, 50 mM Tris, 150 mM β-mercaptoethanol)

• Include protease inhibitors to prevent degradation

• For detection of SMC5/6 complex interactions, consider using Benzonase nuclease to exclude DNA-mediated interactions

Blotting conditions:

• NSMCE3 migrates at approximately 32 kDa on SDS-PAGE gels

• Use 6-12% acrylamide gels for optimal resolution

• Transfer to nitrocellulose membranes shows better results than PVDF for many researchers

Antibody dilutions:

• Primary NSMCE3 antibodies typically work well at 1:1000 dilution

• Secondary antibody dilutions should be optimized based on detection method

Special considerations:

• Patient-derived cell lines with NSMCE3 mutations may show dramatically reduced levels of NSMCE3 and other SMC5/6 complex components (SMC5, SMC6)

• When comparing mutant versus wild-type NSMCE3, inclusion of loading controls is critical as mutations may affect protein stability

How can I use NSMCE3 antibodies for studying protein-protein interactions within the SMC5/6 complex?

NSMCE3 antibodies are valuable tools for investigating protein-protein interactions within the SMC5/6 complex through these methodological approaches:

Co-immunoprecipitation (Co-IP):

• Prepare cell lysates using lysis buffer containing 150 mM NaCl, 50 mM Tris-HCl pH 7.5, 2 mM MgCl₂, 1% NP40, and Benzonase nuclease (90 U/ml) to eliminate DNA-mediated interactions

• Pre-clear lysates by centrifugation at 65,000 × g at 4°C for 30 minutes

• Immunoprecipitate with approximately 5 μg of NSMCE3 antibody and protein A-sepharose beads

• Wash complexes with buffer containing 150 mM NaCl, 50 mM Tris-HCl pH 7.5, 0.5% NP40

• Analyze by SDS-PAGE followed by immunoblotting for other SMC5/6 components

GFP-Trap for tagged proteins:
When using GFP-tagged NSMCE3:

• Transfect cells with GFP-NSMCE3 expression vectors

• Treat cells with DNA damaging agents if studying damage response (e.g., 2 mM hydroxyurea for 16 hours)

• Incubate 3-5 mg of lysate with GFP-Trap agarose beads for 5 hours at 4°C

• Analyze captured complexes for interaction partners

The choice between these methods depends on whether you're studying endogenous or overexpressed NSMCE3. For endogenous studies, antibody quality is critical as non-specific binding can lead to false positives .

What controls should be included when studying NSMCE3 mutations and their effect on the SMC5/6 complex?

When studying NSMCE3 mutations and their impact on the SMC5/6 complex, include these comprehensive controls:

Expression controls:

• Verify equal expression levels of wild-type and mutant NSMCE3 constructs

• Use epitope tags (HA, Myc, or GFP) to confirm expression independent of the protein's structure

• Include mRNA expression analysis to distinguish between transcriptional and post-translational effects

Structural integrity controls:

• Compare recombinant wild-type and mutant NSMCE3 protein migration patterns in size-exclusion chromatography to detect protein folding differences

• Use circular dichroism or thermal shift assays to assess structural stability changes

Interaction controls:

• Include known NSMCE3 interaction-deficient mutants as positive controls (e.g., Leu97Ala for NSMCE1 binding defects and Phe266Ala for NSMCE4 binding defects)

• Perform reciprocal co-immunoprecipitations to confirm interaction changes

• Test interactions with all SMC5/6 components, not just the direct binding partners

Functional assays:

• Assess DNA damage sensitivity (e.g., survival assays with DNA-damaging agents)

• Measure chromosome segregation errors through micronuclei formation assays

• Evaluate homologous recombination efficiency

A comprehensive study on pathogenic NSMCE3 variants demonstrated that mutations (p.Leu264Phe and p.Pro209Leu) abolished binding to NSMCE4 while showing variable effects on NSMCE1 binding. The study validated these findings through multiple approaches including yeast 2-hybrid studies, recombinant protein analysis, and co-immunoprecipitation in human cells .

How do NSMCE3 antibodies help elucidate the role of the SMC5/6 complex in DNA damage response pathways?

NSMCE3 antibodies provide critical insights into SMC5/6 complex function in DNA damage response through these methodological applications:

Temporal dynamics of complex assembly:

• Following DNA damage induction (e.g., ionizing radiation, replication stress agents), use NSMCE3 antibodies in chromatin immunoprecipitation (ChIP) experiments to map recruitment to damage sites

• Combine with PCNA or γH2AX co-staining to determine relationship to replication forks or damage foci

• Use phospho-specific antibodies to detect post-translational modifications of NSMCE3 after damage

Pathway dependency analysis:

• Deplete key DNA damage response factors (ATR, ATM, BRCA1/2) using siRNA or inhibitors

• Examine changes in NSMCE3 localization, complex formation, and post-translational modifications

• Correlate with cellular phenotypes like chromosome breakage and micronuclei formation

Recent research has shown that the SMC5/6 complex plays a critical role in preventing genotoxicity upon APOBEC3A expression, with loss of SMC5/6 components (including NSMCE3) resulting in synthetic lethality with APOBEC3A expression . This finding was validated using functional assays showing that SMC5/6 prevents elongated replication tracts and increases in DNA breaks upon APOBEC3A activity .

Additionally, research has demonstrated that NSMCE3 mutations that destabilize the SMC5/6 complex lead to lung disease-immunodeficiency-chromosomal breakage syndrome (LICS) , highlighting the clinical relevance of SMC5/6 function in maintaining genome stability.

What experimental approaches can resolve contradictions in NSMCE3 antibody-based research findings?

Contradictory findings in NSMCE3 antibody research can be addressed through these methodological approaches:

Antibody validation inconsistencies:

• Perform side-by-side comparisons of commercially available antibodies using the same experimental conditions

• Evaluate antibody specificity through knockdown/knockout controls for each antibody

• Test different fixation and extraction methods that may affect epitope accessibility

Functional discrepancies:

• Use complementary techniques that don't rely solely on antibodies (e.g., CRISPR tagging of endogenous NSMCE3)

• Compare different cell types, as SMC5/6 function may be context-dependent

• Assess the impact of cell cycle synchronization on results, as SMC5/6 functions vary throughout the cell cycle

Interaction contradictions:

• Employ proximity ligation assays (PLA) to confirm protein-protein interactions in situ

• Use mass spectrometry-based approaches like BioID or APEX to map the NSMCE3 interactome under different conditions

• Consider that interactions may be transient or occur only at specific genomic loci

A notable example of resolving contradictions comes from research on the phenotypic differences between mutations in SMC5/6 complex components. Studies have shown that while mutations in both NSMCE2 and NSMCE3 lead to similar cellular phenotypes, their clinical presentations differ significantly . Careful comparative analysis revealed that NSMCE3 mutations destabilize the SMC5/6 complex to a much greater extent than NSMCE2 mutations, potentially explaining the difference in disease manifestations .

How can NSMCE3 antibodies be used to investigate the role of the SMC5/6 complex in viral infections?

Recent research has uncovered important connections between the SMC5/6 complex and viral infections that can be investigated using NSMCE3 antibodies:

Epstein-Barr virus (EBV) interactions:

• Use NSMCE3 antibodies to monitor SMC5/6 complex stability during EBV infection

• Temporal proteomic mapping has shown that multiple SMC5/6 complex subunits, including NSMCE3, are among the most highly depleted human proteins 48 hours after EBV infection

• Investigate protein-protein interactions between viral proteins (e.g., BNRF1) and SMC5/6 components using co-immunoprecipitation with NSMCE3 antibodies

• Assess ubiquitination of SMC5/6 components during viral infection by immunoprecipitating with NSMCE3 antibodies followed by ubiquitin detection

Experimental protocol for detecting SMC5/6 degradation during viral infection:

• Infect relevant cell types (e.g., B cells for EBV studies)

• Harvest cells at various time points post-infection

• Prepare whole-cell lysates and analyze by western blotting for NSMCE3 and other SMC5/6 components

• Include proteasome inhibitors (e.g., bortezomib) or neddylation antagonists (e.g., MLN4924) to determine if viral proteins target SMC5/6 for degradation

• Compare protein levels with mRNA expression to determine if regulation occurs at the post-transcriptional level

Research has shown that BNRF1, an EBV protein, drives calpain- and Cul7-dependent SMC6 turnover, leading to rapid depletion of SMC5/6 components after infection . This example illustrates how NSMCE3 antibodies can be used to understand virus-host interactions that target genome maintenance pathways.

Why might NSMCE3 antibodies show unexpected molecular weight bands, and how should this be interpreted?

When NSMCE3 antibodies detect unexpected molecular weight bands, consider these technical interpretations and solutions:

Common causes and interpretations:

Case example from research:
Studies with Pro209Leu NSMCE3 variant demonstrated that this mutation makes the protein highly prone to C-terminal truncation, resulting in a species migrating with a smaller molecular mass that corresponds to just the N-terminal WH/A subdomain . This finding highlights how mutations can affect protein stability and processing, leading to altered migration patterns on gels.

Methodological approach to resolve issues:

• Include positive controls with recombinant NSMCE3 of known size

• Use epitope-tagged NSMCE3 (N- and C-terminal tags) to distinguish between truncation and modification

• Perform mass spectrometry analysis to confirm the identity of unexpected bands

• Test multiple antibodies targeting different regions of NSMCE3 to confirm specificity

• For patient-derived samples with mutations, compare with wild-type controls to identify mutation-specific banding patterns

What are the best practices for using NSMCE3 antibodies in co-immunoprecipitation studies of the SMC5/6 complex?

For optimal co-immunoprecipitation of NSMCE3 and the SMC5/6 complex, follow these best practices:

Buffer optimization:

• Use lysis buffers that preserve protein-protein interactions while efficiently extracting nuclear proteins

• A recommended buffer composition: 150 mM NaCl, 50 mM Tris-HCl pH 7.5, 2 mM MgCl₂, 1% NP40, with protease inhibitors

• Include Benzonase nuclease (90 U/ml) to eliminate DNA-mediated interactions that may appear as false positives

Pre-clearing strategy:

• Pre-clear lysates by centrifugation at 65,000 × g at 4°C for 30 minutes to remove aggregates

• For sticky proteins, pre-clear with protein A/G beads without antibody

Antibody selection and validation:

• Test multiple NSMCE3 antibodies targeting different epitopes

• Validate specificity through knockdown experiments

• Consider using tagged versions (HA, Myc, GFP) for difficult-to-immunoprecipitate proteins

Detection strategies:

• For low abundance interactions, scale up starting material (3-5 mg of total protein)

• Use appropriate controls (isotype control antibodies, input samples)

• For detecting complex interactions, probe blots for multiple SMC5/6 components

Special considerations for mutant studies:
When studying mutations that affect complex stability, adjust protocols accordingly:

• The Leu264Phe NSMCE3 variant maintains interaction with NSMCE1 but shows reduced incorporation into the native SMC5/6 complex

• For unstable mutants, consider crosslinking approaches to capture transient interactions

• Compare wild-type and mutant interactions under both normal and stress conditions (e.g., after DNA damage)

How can mass spectrometry be combined with NSMCE3 antibodies to study complex composition and dynamics?

Integrating mass spectrometry with NSMCE3 immunoprecipitation provides powerful insights into complex composition and dynamics:

Sample preparation protocols:

• Scale up immunoprecipitation using NSMCE3 antibodies (aim for at least 10 mg of starting material)

• Perform on-bead digestion with trypsin directly after immunoprecipitation

• For quantitative analysis, consider SILAC labeling of cells before immunoprecipitation

  • Maintain cells in RPMI without L-lysine and L-arginine supplemented with isotope-labeled amino acids (e.g., ¹³C₆, ¹⁵N₂-L-lysine)

  • For control samples, use regular amino acids

• For comparing wild-type vs. mutant NSMCE3, label each condition differently

Data analysis approach:

• Calculate dependences of mitotic chromosomal-associating proteins using SILAC ratios averaged across experiments

• Apply advanced analysis tools like NanoRF (based on Random Forests algorithm) for supervised classification

• Replace missing values with median SILAC values and assess classification quality using ROC-curve analysis and Matthews correlation coefficient

Experimental design considerations:

• Include appropriate controls (IgG immunoprecipitation, knockdown samples)

• Perform biological replicates (minimum of three) with label swapping in SILAC experiments

• For temporal dynamics, collect samples at multiple time points after stimulus (e.g., DNA damage)

Research has demonstrated the power of this approach in analyzing the proteomics of SMC complex components. For example, a study using a nano Random Forest approach revealed novel insights into the chromosome-associated proteome after depletion of various SMC components . Each sample for mass spectrometry analysis was generated by combining three individual preparations, enhancing reliability and reproducibility .

How might new NSMCE3 antibody technologies advance our understanding of SMC5/6 complex dynamics in live cells?

Emerging antibody technologies offer exciting prospects for studying NSMCE3 and SMC5/6 complex dynamics:

Single-domain antibodies (nanobodies):

• Develop anti-NSMCE3 nanobodies for live-cell imaging

• Express fluorescently tagged nanobodies in cells to track NSMCE3 localization during DNA damage response and mitosis

• Advantage: Smaller size allows access to dense chromatin regions and nuclear pores

Antibody-based proximity labeling:

• Conjugate NSMCE3 antibodies to enzymes like APEX2 or TurboID for proximity labeling

• Use cell-permeable antibody delivery methods to label NSMCE3 proximity partners in living cells

• Apply this technique to map differential SMC5/6 complex composition at specific genomic loci

Split-fluorescent protein complementation:

• Generate cell lines expressing NSMCE3 fused to one half of a split fluorescent protein

• Express potential interaction partners fused to the complementary half

• Use this system to visualize real-time complex assembly and disassembly after DNA damage

High-resolution imaging applications:

• Develop super-resolution-compatible NSMCE3 antibodies for techniques like STORM or PALM

• Use multi-color imaging to simultaneously track multiple SMC5/6 components

• Combine with chromatin imaging to correlate with DNA replication or repair sites

These advanced approaches could resolve fundamental questions about SMC5/6 function, such as whether complex composition changes in response to different types of DNA damage or across different cell cycle phases.

What role might NSMCE3 antibodies play in understanding the connection between the SMC5/6 complex and neurodevelopmental disorders?

NSMCE3 antibodies could provide critical insights into the emerging connection between SMC5/6 and neurodevelopment:

Brain-specific expression and localization studies:

• Use NSMCE3 antibodies for immunohistochemistry of brain tissues from different developmental stages

• Compare expression patterns in different neural cell types (neurons vs. glia)

• Correlate with markers of neural progenitor proliferation and differentiation

Patient-derived cell models:

• Apply NSMCE3 antibodies to study protein levels and complex formation in induced pluripotent stem cells (iPSCs) from patients with relevant disorders

• Track NSMCE3 localization during neural differentiation in patient vs. control cells

• Assess DNA damage accumulation in developing neurons using NSMCE3 co-localization with γH2AX

Mechanistic investigations:

• Determine if NSMCE3 and the SMC5/6 complex interact with neurodevelopmental transcription factors

• Investigate whether NSMCE3 has brain-specific interaction partners

• Examine the impact of neurodevelopmental disorder-associated mutations on NSMCE3 function

The potential connection to neurodevelopment is supported by the inclusion of NSMCE3 in the Autism research panel , suggesting a possible role in neurodevelopmental disorders. Additionally, NSMCE3's function in maintaining genome stability is particularly crucial in rapidly dividing neural progenitors where DNA replication stress can have profound developmental consequences.

How can NSMCE3 antibodies facilitate translational research connecting basic SMC5/6 biology to immunodeficiency disorders?

NSMCE3 antibodies serve as valuable tools connecting basic research to clinical applications in immunodeficiency:

Diagnostic development:

• Use NSMCE3 antibodies to develop screening assays for SMC5/6 complex deficiency

• Establish immunoblotting protocols that can distinguish between normal and pathologically reduced NSMCE3 levels

• Validate in patient cohorts with undiagnosed primary immunodeficiencies

Therapeutic response biomarkers:

• Monitor NSMCE3 levels and complex formation in patient samples before and after treatments

• Correlate changes in SMC5/6 complex stability with clinical response

• Develop standardized protocols for clinical laboratories

Genotype-phenotype correlation studies:

• Use NSMCE3 antibodies to characterize the molecular consequences of different NSMCE3 variants

• Establish how different mutations affect protein stability, localization, and complex formation

• Correlate biochemical findings with clinical presentations

Current research has established that NSMCE3 mutations cause lung disease immunodeficiency and chromosome breakage syndrome (LICS), characterized by severe pulmonary disease and immunodeficiency in early childhood . The Blue Print Genetics Primary Immunodeficiency Panel includes assessment of NSMCE3 as part of a 336-gene panel for patients with suspected primary immunodeficiency .

Furthermore, NSMCE3 has been included in COVID-19 research panels , suggesting potential connections between SMC5/6 function and viral susceptibility. The Common Variable Immunodeficiency Panel from Invitae also analyzes genes associated with immunodeficiency, hypogammaglobulinemia, and recurrent infections , which overlaps with LICS symptoms.