NCAPD3 Antibody

What is NCAPD3 and what are its primary cellular functions?

NCAPD3 is a subunit of the condensin II complex that plays essential roles in mitotic chromosome assembly and segregation. Condensin complexes contain two invariant structural maintenance of chromosome (SMC) subunits, SMC2 and SMC4, but differ in their non-SMC subunits. NCAPD3 is one of three non-SMC subunits that specifically define condensin II .

Beyond chromosome condensation, NCAPD3 has been found to play key roles in:

• Innate immunity by promoting the formation of interferon activated translation inhibitor (GAIT) complex

• Regulating transcription of genes encoding amino acid transporters (SLC7A5 and SLC3A2)

• Glucose metabolism reprogramming in cancer cells

• Enhancing the Warburg effect in colorectal cancer progression

These diverse functions highlight NCAPD3's significance beyond its classical role in chromosome dynamics.

What applications are validated for NCAPD3 antibodies in research?

NCAPD3 antibodies have been validated for multiple applications with specific dilution recommendations:

These applications are supported by published literature, with Western blotting being the most frequently utilized technique (5 publications referenced) .

How should NCAPD3 antibodies be stored and handled for optimal results?

For optimal results, NCAPD3 antibodies should be stored at -20°C where they remain stable for one year after shipment. The commercial antibody preparations typically come in liquid form, suspended in PBS with 0.02% sodium azide and 50% glycerol at pH 7.3. Notably, aliquoting is indicated as unnecessary for -20°C storage for some preparations. Some formulations (20μl sizes) contain 0.1% BSA as a stabilizer .

Proper handling includes avoiding repeated freeze-thaw cycles and maintaining cold chain during experimental procedures. When designing experiments, titration of the antibody is recommended in each testing system to obtain optimal results, as optimal dilutions may be sample-dependent .

How can researchers validate NCAPD3 antibody specificity in their experimental systems?

Validating NCAPD3 antibody specificity requires a multi-faceted approach:

• Positive and negative controls: Use cell lines with known NCAPD3 expression levels, such as HepG2, HeLa, and MCF-7 cells, which have been verified to express detectable levels of NCAPD3. For negative controls, employ:

  • NCAPD3 knockdown cells using specific shRNAs (as demonstrated in AGS and MGC803 cells)

  • NCAPD3 knockout cells generated by CRISPR/Cas9 technology (as referenced in AGS cells)

• Molecular weight verification: Confirm that the detected band aligns with the expected molecular weight (calculated: 169 kDa; observed: 165 kDa)

• Immunoprecipitation validation: Perform IP followed by Western blot to verify antibody-antigen interaction specificity

• Cross-reactivity assessment: While NCAPD3 antibodies have demonstrated reactivity with human samples, some have cited reactivity with bovine samples as well. Researchers should verify cross-reactivity if working with non-human models

These validation approaches ensure experimental results attributable to NCAPD3 are not artifacts of non-specific antibody binding.

What are the optimal experimental designs for studying NCAPD3 interactions with c-Myc and E2F1?

Research has revealed that NCAPD3 interacts with both c-Myc and E2F1 transcription factors to regulate metabolic pathways in cancer. To study these interactions effectively:

• Co-immunoprecipitation (Co-IP): Use anti-NCAPD3 antibodies to pull down protein complexes, followed by western blotting with c-Myc and E2F1 antibodies. This approach has demonstrated dose-dependent interactions between NCAPD3 and these transcription factors

• Chromatin immunoprecipitation (ChIP): Design experiments to evaluate how NCAPD3 influences c-Myc and E2F1 recruitment to target gene promoters:

  • For c-Myc-regulated genes: GLUT1, HK2, ENO1, PKM2, and LDHA promoters

  • For E2F1-regulated genes: PDK1 and PDK3 promoters

• Sequential ChIP (Re-ChIP): To confirm NCAPD3, c-Myc, and E2F1 co-occupy specific promoters

• Expression correlation studies: Conduct experiments that manipulate NCAPD3 levels (overexpression and knockdown) and measure corresponding changes in c-Myc and E2F1 levels, as research has shown NCAPD3 upregulates both factors

• Functional rescue experiments: In NCAPD3 knockdown models, test whether overexpression of c-Myc or E2F1 can rescue the phenotype

These approaches provide mechanistic insights into how NCAPD3 influences transcriptional networks controlling metabolism.

What methodological considerations are important when using NCAPD3 antibodies for cell cycle studies?

When designing experiments to study NCAPD3 in the context of cell cycle progression:

• Cell synchronization: Since condensin II (containing NCAPD3) shows distinct localization patterns during the cell cycle, researchers should implement appropriate synchronization protocols:

  • NCAPD3 is nuclear during interphase

  • It binds to chromatin during early prophase and remains bound until the end of telophase

  • Different from condensin I, which is cytoplasmic during interphase and binds chromatin after nuclear envelope breakdown

• Co-staining approaches: Combine NCAPD3 immunostaining with cell cycle phase markers:

  • Nuclear envelope markers to distinguish prophase from prometaphase

  • Histone H3 phosphorylation (Ser10) for mitotic cells

  • DAPI for DNA visualization and mitotic figure identification

• Dynamic analysis considerations: Since NCAPD3 plays a role in early chromosome condensation, time-lapse microscopy with fluorescently tagged NCAPD3 may be valuable for studying its dynamics

• Histone mark correlation: Consider co-staining for mono-methyl histone H4 Lys20, as NCAPD3 contains HEAT repeat clusters that bind to this histone mark, which is prevalent during mitosis

• Knockdown timing optimization: When designing NCAPD3 depletion experiments, consider that complete depletion may cause severe mitotic defects, potentially complicating interpretation. Partial or inducible depletion systems may be preferable.

These methodological considerations will enhance the quality and interpretability of cell cycle studies involving NCAPD3.

How does NCAPD3 expression correlate with cancer progression and prognosis?

Multiple studies have demonstrated NCAPD3's significance in cancer:

These findings collectively establish NCAPD3 as a potential biomarker and therapeutic target across multiple cancer types.

What mechanisms underlie NCAPD3's role in promoting the Warburg effect in cancer cells?

NCAPD3 promotes the Warburg effect in cancer cells through several interconnected mechanisms:

• c-Myc-dependent glycolytic enzyme regulation:

  • NCAPD3 upregulates c-Myc levels in cancer cells

  • NCAPD3 interacts with c-Myc and enhances its recruitment to promoters of key glycolytic genes:

    • GLUT1 (glucose transporter)

    • HK2 (hexokinase 2)

    • ENO1 (enolase 1)

    • PKM2 (pyruvate kinase M2)

    • LDHA (lactate dehydrogenase A)

• E2F1-mediated TCA cycle inhibition:

  • NCAPD3 increases E2F1 levels and interacts with E2F1

  • This interaction enhances E2F1 recruitment to promoters of PDK1 and PDK3 genes

  • Increased PDK1/3 expression leads to:

    • Enhanced phosphorylation of PDHE1α at Ser293

    • Inactivation of the pyruvate dehydrogenase complex

    • Reduced conversion of pyruvate to acetyl-CoA

    • Suppression of the TCA cycle

• Metabolic consequence verification:

  • NCAPD3 knockdown decreased PDK1, PDK3, and p-PDHE1α levels

  • Conversely, NCAPD3 overexpression increased these markers

  • The total protein level of PDHE1α remained unchanged, indicating regulation at the post-translational level

This dual mechanism of enhancing glycolysis while suppressing the TCA cycle provides comprehensive reprogramming of glucose metabolism, reinforcing the Warburg effect in cancer cells.

What signaling pathways are affected by NCAPD3 knockdown in cancer cells?

Gene Set Enrichment Analysis (GSEA) following NCAPD3 knockdown revealed several affected signaling pathways and biological processes:

• Canonical pathways:

  • Signaling by receptor tyrosine kinases

  • Cytokine signaling in the immune system

  • Cytokine-cytokine receptor interaction

  • Eight other significantly enriched pathways

• Oncogenic signatures:

  • 26 significantly enriched oncogene sets were identified

  • Top five significantly enriched gene sets include ALK, RB, BMI1, P53, and MEK pathways

• Transcription factor networks:

  • 13 significantly enriched transcription factor gene sets

  • Most significant include P53, FOXO4, and FOXO1

• Cellular components:

  • Five significant enrichment results in cellular component GSEA

  • Cell surface gene sets were significantly enriched

• Immunologic signatures:

  • 119 immune gene sets were significantly enriched following NCAPD3 knockdown

  • Consistent with canonical pathway enrichment showing cytokine signaling activation

These findings suggest NCAPD3 functions as a master regulator affecting multiple signaling networks and biological processes beyond its classical role in chromosome biology.

How can researchers optimize Western blotting protocols for NCAPD3 detection?

Given NCAPD3's high molecular weight (observed: 165 kDa), several optimization strategies are recommended:

• Gel electrophoresis optimization:

  • Use low percentage gels (6-8%) to allow better separation of high molecular weight proteins

  • Extend running time for adequate separation of large proteins

  • Consider gradient gels for improved resolution

• Transfer optimization:

  • Employ wet transfer rather than semi-dry methods for large proteins

  • Use lower voltage for longer periods (e.g., 30V overnight at 4°C)

  • Add SDS (0.1%) to transfer buffer to facilitate large protein transfer

  • Consider specialized transfer methods for high molecular weight proteins

• Antibody incubation:

  • Use validated dilutions (1:500-1:1000) as recommended in product specifications

  • Consider longer primary antibody incubation times (overnight at 4°C)

  • Optimize blocking solutions to minimize background while preserving specific signal

• Positive controls:

  • Include lysates from cells with known NCAPD3 expression (HepG2, HeLa, MCF-7)

  • Consider including both positive and negative controls on the same blot

• Detection system selection:

  • Enhanced chemiluminescence (ECL) systems with increased sensitivity are recommended

  • Optimize exposure times, as NCAPD3 may require longer exposures for clear visualization

These optimizations account for the challenges inherent in detecting large proteins like NCAPD3 and will increase the likelihood of successful Western blotting experiments.

What experimental controls should be included when studying NCAPD3 function in cell models?

Robust experimental design for NCAPD3 functional studies should include:

• Expression manipulation controls:

  • Confirmation of knockdown/overexpression efficiency by both RT-qPCR and Western blotting

  • Multiple shRNA sequences to control for off-target effects

  • Scrambled/non-targeting shRNA or empty vector controls

  • Rescue experiments with shRNA-resistant NCAPD3 constructs to confirm specificity

• Cellular phenotype controls:

  • Positive controls for assays (proliferation, apoptosis, invasion, migration)

  • Time-course analyses to distinguish immediate from secondary effects

  • Multiple cell lines to ensure findings aren't cell-type specific

• Mechanistic investigation controls:

  • When studying NCAPD3's interaction with c-Myc and E2F1:

    • Include IgG controls for co-immunoprecipitation

    • Perform reciprocal IP (pull down with c-Myc or E2F1 antibodies, blot for NCAPD3)

    • Include controls for binding specificity (e.g., other transcription factors)

• In vivo model controls:

  • Studies using AOM/DSS-induced colorectal cancer models should include:

    • Wild-type controls

    • Heterozygous controls (NCAPD3+/-)

    • Appropriate timing controls for AOM/DSS administration

• Pathway verification controls:

  • When studying metabolic effects:

    • Include metabolic inhibitor controls

    • Measure multiple parameters of glycolysis/TCA cycle

    • Compare results with known glycolytic regulators

These comprehensive controls enhance data reliability and facilitate interpretation of complex phenotypes resulting from NCAPD3 manipulation.

What considerations are important for immunofluorescence studies of NCAPD3 in different cell cycle phases?

NCAPD3 localization changes throughout the cell cycle, requiring specific considerations for immunofluorescence studies:

• Fixation and permeabilization optimization:

  • Compare different fixation methods (paraformaldehyde, methanol, or combination)

  • Test various permeabilization agents (Triton X-100, saponin) at different concentrations

  • Optimize fixation duration to preserve NCAPD3 epitopes while maintaining cellular architecture

• Antibody dilution and incubation:

  • Test the recommended range (1:50-1:500) to determine optimal signal-to-noise ratio

  • Consider extended primary antibody incubation (overnight at 4°C)

  • Include peptide competition controls to confirm specificity

• Co-localization studies:

  • Include markers for nuclear envelope (e.g., lamin B1) to distinguish interphase from prophase

  • Co-stain with chromatin markers (e.g., DAPI) to visualize chromosomal association

  • Consider co-staining with mono-methyl histone H4 Lys20, a binding partner for NCAPD3

• Cell cycle phase identification:

  • Use synchronized cell populations or cell cycle markers to identify specific phases

  • Remember that NCAPD3 (condensin II) is nuclear during interphase and binds chromatin in early prophase

  • This differs from condensin I, which is cytoplasmic during interphase

• Imaging considerations:

  • Use confocal microscopy for precise localization studies

  • Employ z-stack imaging to capture the full nuclear volume

  • Consider super-resolution microscopy for detailed chromatin association studies

These guidelines will help researchers obtain clear, interpretable results when studying NCAPD3's dynamic localization throughout the cell cycle.

What are promising therapeutic strategies targeting NCAPD3 in cancer?

Based on current understanding, several therapeutic approaches targeting NCAPD3 show promise:

• Direct inhibition strategies:

  • Development of small molecule inhibitors targeting NCAPD3's HEAT repeat domains

  • Disruption of NCAPD3's protein-protein interactions, particularly with c-Myc and E2F1

  • Peptide-based inhibitors mimicking key interaction interfaces

• Transcriptional/translational targeting:

  • siRNA/shRNA delivery systems for NCAPD3 silencing, supported by evidence that:

    • NCAPD3 silencing attenuated malignant phenotypes of gastric cancer cells

    • NCAPD3 knockout suppressed colorectal cancer development in mouse models

  • Antisense oligonucleotides targeting NCAPD3 mRNA

  • CRISPR-based approaches for precision editing of NCAPD3 regulatory elements

• Metabolic intervention strategies:

  • Combination approaches targeting both NCAPD3 and glycolytic enzymes

  • PDK inhibitors to counteract NCAPD3-mediated PDK upregulation

  • Metabolic reprogramming therapies to reverse the Warburg effect

• Synthetic lethality approaches:

  • Identification of genetic dependencies in NCAPD3-overexpressing cancers

  • Exploration of potential vulnerabilities in chromosome segregation pathways

  • Combination with cell cycle checkpoint inhibitors

• Biomarker-guided therapy selection:

  • Development of diagnostics to identify patients with NCAPD3-driven tumors

  • Patient stratification based on NCAPD3 expression levels

  • Monitoring NCAPD3 as a resistance biomarker

The convergence of NCAPD3's roles in chromosome biology and metabolic regulation offers multiple intervention points, with particular promise in combination therapeutic strategies.

How might NCAPD3 function differ across cancer types, and what methodological approaches can address this question?

Evidence suggests NCAPD3 may have context-dependent functions across cancer types:

• Multi-cancer comparative analysis approaches:

  • Systematic immunohistochemistry across cancer tissue microarrays

  • Pan-cancer NCAPD3 expression correlation with clinical outcomes

  • Comparison of subcellular localization patterns across tumor types

• Mechanistic investigation methodologies:

  • ChIP-seq in multiple cancer cell lines to identify common and unique NCAPD3 binding sites

  • Proteomics-based identification of NCAPD3 interaction partners across cancer types

  • Transcriptome analysis following NCAPD3 manipulation in diverse cancer models

• Functional conservation assessment:

  • Standardized functional assays (proliferation, apoptosis, migration) following NCAPD3 knockdown

  • Cross-cancer metabolic profiling to determine if the Warburg effect enhancement is universal

  • Rescue experiments to test if NCAPD3 from one cancer type can restore function in another

• Genetic context considerations:

  • Analysis of NCAPD3 mutations across cancer types and their functional consequences

  • Investigation of genetic modifiers that influence NCAPD3 function

  • Exploration of cancer-specific post-translational modifications

• Translational approaches:

  • Development of cancer-type specific biomarker panels including NCAPD3

  • Assessment of NCAPD3 as a therapeutic target across cancer types

  • Identification of cancer contexts where NCAPD3-targeting would be most effective

These methodological approaches would help determine whether NCAPD3 represents a broadly applicable cancer target or if its utility is limited to specific cancer contexts.

What is the relationship between NCAPD3's canonical role in chromosome biology and its emerging functions in metabolism?

The intersection between NCAPD3's chromosome biology and metabolic functions represents an intriguing research frontier:

• Mechanistic integration hypotheses:

  • NCAPD3 may serve as a sensor linking cell cycle progression to metabolic demands

  • Its chromosome-associated and transcription factor-associated pools may be distinctly regulated

  • Potential for cell cycle-dependent regulation of NCAPD3's metabolic functions

• Experimental approaches to address this relationship:

  • Cell cycle synchronization followed by analysis of NCAPD3's metabolic functions

  • Development of separation-of-function mutants that distinguish chromosomal from metabolic roles

  • Chromatin fractionation to determine if metabolism-regulating NCAPD3 is chromatin-bound

• Evolutionary perspective investigations:

  • Comparative analysis of NCAPD3 functions across species

  • Identification of conserved domains mediating different functions

  • Exploration of when these dual functions emerged evolutionarily

• Integrative multi-omics approaches:

  • Combined analysis of NCAPD3 genomic binding (ChIP-seq), transcriptome effects (RNA-seq), and metabolomic consequences

  • Proteomic identification of post-translational modifications that might switch NCAPD3 between functions

  • Spatial proteomics to map NCAPD3 interactomes in different cellular compartments

• Disease-relevant contexts:

  • Investigation of how cancer mutations in NCAPD3 affect both chromosome biology and metabolism

  • Analysis of whether metabolic stress alters NCAPD3's chromosome functions

  • Exploration of potential feedback loops between these processes