ZC3HC1 Antibody

What is ZC3HC1 and why is it an important research target?

ZC3HC1 (zinc finger C3HC-type protein 1), also known as Nuclear-interacting partner of ALK (NIPA), is a multi-functional protein with significant roles in cellular processes. Recent research has redefined ZC3HC1 as a structural component of the nuclear basket (NB), a fibrillar structure attached to the nuclear pore complex (NPC) . Earlier studies characterized it as an F-box-containing protein that functions within SCF-type E3 ubiquitin ligase complexes regulating cell cycle progression .

The significance of ZC3HC1 as a research target has increased with discoveries about its:

• Role in nuclear basket architecture and nuclear pore complex function

• Involvement in cell cycle regulation, particularly the G2/M transition

• Associations with disease states, including SNPs linked to coronary artery disease and altered expression in certain cancers

This dual functionality as both a structural nuclear component and cell cycle regulator makes ZC3HC1 antibodies valuable tools for investigating nuclear envelope dynamics, cell cycle control mechanisms, and related pathologies.

How should I design experiments to distinguish between the nuclear basket and SCF complex functions of ZC3HC1?

Distinguishing between ZC3HC1's roles requires careful experimental design that can differentiate its localization and interaction partners:

Methodological approach:

• Subcellular fractionation with immunoblotting:

  • Perform nuclear envelope isolation alongside cytoplasmic and nuclear soluble fractions

  • Compare ZC3HC1 distribution across fractions using validated antibodies

  • Reference study: Gunkel et al. (2021) demonstrated that in Xenopus laevis cells (XL-177), "the largest amount of ZC3HC1 within this cell type was nonetheless clearly detectable within its LNN-enriched fractions" while "certain amounts of ZC3HC1 exist in a soluble form in interphase"

• Co-immunoprecipitation (Co-IP) assays:

  • Perform parallel Co-IPs targeting:

    • Nuclear basket proteins (TPR) to capture NB-associated ZC3HC1

    • SCF components (SKP1, CUL1) to capture E3 ligase-associated ZC3HC1

  • Quantify relative distribution between complexes

• Microscopy-based approaches:

  • Implement dual-color immunofluorescence for ZC3HC1 alongside:

    • Nuclear basket markers (TPR)

    • SCF complex components

  • Analyze co-localization coefficients quantitatively

• Functional validation through domain-specific mutations:

  • The study by Gunkel describes a "bimodular NuBaID" (nuclear basket-interaction domain) in ZC3HC1 essential for TPR binding

  • Design constructs with mutations in:

    • The NuBaID to disrupt nuclear basket association

    • The F-box domain to disrupt SCF complex formation

  • Express these in ZC3HC1 knockout cells to assess function-specific rescue

This combination of approaches allows for robust differentiation between ZC3HC1's dual roles and provides framework for investigating function-specific perturbations.

What controls should be included when validating a new ZC3HC1 antibody for research applications?

A comprehensive validation strategy for ZC3HC1 antibodies should include the following controls:

• Specificity controls:

  • Knockout validation: Test antibody in ZC3HC1 knockout cells (created via CRISPR/Cas9 technology as described in Gunkel et al.)

  • Knockdown validation: Compare signal in cells treated with siRNA targeting ZC3HC1 versus non-targeting control

  • Overexpression validation: Test in cells overexpressing tagged ZC3HC1 to confirm co-localization

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

• Technical controls:

  • Loading controls: Include appropriate housekeeping proteins (e.g., GAPDH for Western blot)

  • Secondary-only controls: Omit primary antibody to assess background from secondary detection

  • Isotype controls: Use matched isotype antibodies to determine non-specific binding

• Application-specific validation:

  • Western blot: Confirm detection at the expected molecular weight (~55 kDa)

  • Immunofluorescence: Verify nuclear envelope rim staining consistent with nuclear basket localization

  • Immunohistochemistry: Compare staining pattern across multiple tissue types known to express ZC3HC1

• Cross-validation:

  • Compare results with at least two independent antibodies targeting different epitopes

  • Compare antibody performance against recombinant ZC3HC1 fusion proteins

Successful validation would demonstrate absence of signal in knockout/knockdown conditions, consistent molecular weight detection, expected subcellular localization, and reproducibility across different antibody clones targeting the same protein.

How can I examine the interaction between ZC3HC1 and TPR proteins in the nuclear basket structure?

Investigating ZC3HC1-TPR interactions requires specialized techniques that can detect protein-protein interactions at the nuclear basket. Based on published methodologies:

• Proximity ligation assay (PLA):

  • This technique can visualize protein interactions within 40nm distance in situ

  • Fix cells and probe with primary antibodies against ZC3HC1 and TPR

  • Use secondary antibodies conjugated with oligonucleotides

  • Ligase and polymerase reactions create amplified fluorescent signals at interaction sites

  • Quantify PLA signals at the nuclear envelope

• FRET-based approaches:

  • Express fluorescently-tagged ZC3HC1 and TPR constructs

  • Measure Förster resonance energy transfer at the nuclear envelope

  • This approach was referenced in research showing that "fluorescence-loss-in-photobleaching (FLIP) experiments in HeLa cells had revealed that such interactions between FP-tagged ZC3HC1 and the NBs lasted far longer than those between the NPCs and transiently interacting proteins"

• Yeast two-hybrid (Y2H) analysis:

  • The research by Gunkel et al. used Y2H to demonstrate that "all ZC3HC1 mutants capable of NB binding also showed a robust Y2H interaction with TPR"

  • Design constructs with specific domains of each protein

  • Test domain-specific interactions to map binding interfaces

• Co-immunoprecipitation with domain mapping:

  • Use antibodies against either ZC3HC1 or TPR to pull down protein complexes

  • Identify interacting domains through truncation or mutation analysis

  • Research demonstrated that "ZC3HC1 uses the tandem arrangement of two BLDs for its binding to the NB, with the functionality of both domains depending on residues likely to be involved in zinc ion coordination"

• Super-resolution microscopy:

  • Techniques such as STORM or PALM can resolve proteins at nanometer resolution

  • Visualize the spatial organization of ZC3HC1 and TPR at the nuclear basket

  • Quantify co-localization with precision beyond conventional microscopy

These approaches provide complementary data on different aspects of the ZC3HC1-TPR interaction, from biochemical binding to spatial organization at the nuclear envelope.

What approaches can resolve contradictory findings about ZC3HC1 expression in proliferating versus non-dividing cells?

Earlier literature described ZC3HC1/NIPA as "occurring only in minimal amounts in growth-arrested cells" , while more recent findings suggest it's present in both proliferating and non-dividing cells. To resolve these contradictions:

• Cell cycle-synchronized analysis:

  • Synchronize cells at different cell cycle stages using established methods (double thymidine block, nocodazole arrest, serum starvation)

  • Quantify ZC3HC1 levels by Western blot and immunofluorescence across all cell cycle phases

  • Include both nuclear envelope fraction and total cellular protein

  • Compare results across multiple antibodies targeting different epitopes

• Comparative tissue analysis:

  • Analyze ZC3HC1 expression in:

    • Highly proliferative tissues (intestinal epithelium, skin)

    • Post-mitotic tissues (adult neurons, cardiomyocytes)

    • Quiescent tissues (G0 arrested cells)

  • Use both quantitative proteomics and immunohistochemistry

• Distinguish protein pools and modifications:

  • Perform phosphorylation-specific analysis (described in Gunkel et al. for Xenopus egg extracts )

  • Separate nuclear envelope-bound versus soluble ZC3HC1

  • Research indicates ZC3HC1 is "common at the nuclear envelopes (NE) of proliferating and non-dividing, terminally differentiated cells of different morphogenetic origin"

• Multi-omics approach:

  • Compare transcriptomics, proteomics, and localization data

  • Correlate with cell proliferation markers

  • Look for post-translational modifications that might affect detection or function

• Analysis of different ZC3HC1 pools:

  • The study by Gunkel et al. found that "in some [cell types] ZC3HC1 was even located exclusively at the NE and only detectable within the LNN-enriched materials"

  • Compare different extraction methods to determine if methodological differences explain contradictory findings

This comprehensive approach should reveal whether the contradictions stem from technical limitations, differential regulation of distinct ZC3HC1 pools, or cell type-specific expression patterns.

What are the optimal conditions for using ZC3HC1 antibodies in Western blotting applications?

Based on validated protocols and research publications, these are the optimized conditions for Western blotting with ZC3HC1 antibodies:

Sample preparation:

• Include phosphatase inhibitors in lysis buffer as ZC3HC1 is subject to phosphorylation

• For complete extraction, use buffers containing non-ionic detergents (e.g., NP-40 or Triton X-100)

• For nuclear envelope enrichment, consider subcellular fractionation protocols

SDS-PAGE conditions:

• Use 10-12% polyacrylamide gels for optimal resolution around 55 kDa

• Load 20-40 μg of total protein per lane (cell line-dependent)

• Include positive control lysates (A549 cells, mouse tissues)

Transfer and blocking:

• Standard wet transfer to nitrocellulose membranes

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

Antibody incubation:

• Primary antibody dilution: 1:500-1:1000 in blocking buffer

• Incubate overnight at 4°C with gentle agitation

• Secondary antibody: HRP-conjugated anti-rabbit IgG at 1:5000-1:10000

• Expected band: ~55 kDa

Detection and validation:

• Use enhanced chemiluminescence (ECL) detection systems

• Include molecular weight markers spanning 40-70 kDa range

• Verify specificity using ZC3HC1 knockout controls when available

Troubleshooting:

• If multiple bands appear, optimize primary antibody concentration

• For weak signals, extend primary antibody incubation time or increase protein loading

• For high background, increase washing steps or reduce antibody concentration

These conditions have been validated in multiple research contexts and provide a starting point for reliable ZC3HC1 detection.

How can I optimize immunofluorescence staining protocols for ZC3HC1 at the nuclear envelope?

Optimizing immunofluorescence for ZC3HC1 at the nuclear envelope requires specific attention to fixation, permeabilization, and imaging parameters:

Fixation optimization:

• Test multiple fixation methods:

  • 4% paraformaldehyde (10-15 minutes at room temperature)

  • Methanol fixation (-20°C for 10 minutes)

  • Combined fixation (PFA followed by methanol)

• Research indicates that preservation of nuclear envelope structures may require specific fixation protocols to maintain nuclear basket integrity

Permeabilization considerations:

• Nuclear envelope proteins require balanced permeabilization:

  • Too harsh: may extract nuclear basket components

  • Too mild: may prevent antibody access

• Recommended starting protocol:

  • 0.2% Triton X-100 for 10 minutes post-fixation

  • Alternative: 0.05% saponin for more gentle permeabilization

Blocking and antibody conditions:

• Block with 5% normal serum (matching secondary antibody host) with 0.1% Triton X-100

• Primary antibody: Use at 1:200-1:500 dilution

• Incubate overnight at 4°C in humidified chamber

• Include nuclear pore marker (e.g., TPR) for co-localization studies

Signal enhancement strategies:

• Consider tyramide signal amplification for weak signals

• Use high-sensitivity detection systems (quantum dots, Alexa Fluor dyes)

• For co-localization with TPR, ensure spectral separation between fluorophores

Imaging considerations:

• Super-resolution microscopy (SIM, STED) provides superior visualization of nuclear envelope structures

• Confocal microscopy with Airyscan or similar technology improves resolution

• Capture z-stacks with 0.2-0.3 μm steps to fully resolve the nuclear envelope

Validation approaches:

• Include ZC3HC1 knockout cells as negative controls

• Compare with tagged ZC3HC1 expression patterns

• Use multiple antibodies targeting different epitopes

These optimizations should enable clear visualization of ZC3HC1 at the nuclear envelope with minimal background and high specificity.

How can I investigate the functional significance of the bimodular nuclear basket-interaction domain (NuBaID) in ZC3HC1?

The discovery of the bimodular NuBaID in ZC3HC1 provides opportunities for sophisticated functional analysis. Based on research by Gunkel et al. , these approaches can elucidate its significance:

• Structure-function analysis using domain mutants:

  • Generate point mutations in key residues of both NuBaID modules (BLD1 and BLD2)

  • Research identified critical residues: "the impaired NB binding of a ZC3HC1 mutant reflected its impaired interaction with TPR"

  • Express these mutants in ZC3HC1 knockout cells

  • Assess:

    • Nuclear basket localization

    • TPR binding capacity

    • Functional complementation

• Domain replacement experiments:

  • Create chimeric proteins by replacing ZC3HC1's NuBaID with:

    • Corresponding domains from evolutionary homologs (e.g., DDB0349234 from D. discoideum or Pml39p from S. cerevisiae)

    • Synthetic modules designed to mimic NuBaID structure

  • Test their ability to localize to nuclear baskets and interact with TPR

• Structural biology approaches:

  • Utilize computational predictions like those from AlphaFold2

  • Conduct experimental structure determination of the NuBaID

  • Map interaction interfaces with TPR through cross-linking mass spectrometry

• Evolutionary analysis:

  • Compare NuBaID function across diverse eukaryotic lineages

  • Research indicates "each ZC3HC1-positive species with a nonduplicated genome... found only one NuBaID signature–containing protein"

  • Assess co-evolution with TPR homologs

  • Identify conserved and divergent features

• Functional assays:

  • Analyze nuclear transport efficiency in cells expressing NuBaID mutants

  • Examine gene expression changes when NuBaID function is disrupted

  • Assess cell cycle progression in cells with NuBaID mutations

  • Measure nuclear envelope stability and nuclear pore complex distribution

These approaches provide comprehensive insights into how the bimodular NuBaID enables ZC3HC1's structural role at the nuclear basket and its functional consequences for cellular processes.

What methodologies can determine if the E3 ubiquitin ligase function of ZC3HC1 is related to or independent from its nuclear basket role?

This fundamental question requires sophisticated experimental design to untangle potentially connected or independent functions:

• Domain-specific functional uncoupling:

  • Generate domain-specific ZC3HC1 mutants:

    • NuBaID mutants: disrupt nuclear basket binding while preserving F-box domain

    • F-box mutants: disrupt SCF complex formation while preserving nuclear basket binding

  • Express these in ZC3HC1 knockout cells

  • Assess rescue of function for both activities independently

• Proximity-dependent labeling:

  • Employ BioID or TurboID fusions with ZC3HC1

  • Map the proximal interactome in different cellular compartments

  • Identify proteins that interact with ZC3HC1 in:

    • Nuclear basket context

    • SCF complex context

  • Compare interactome overlap and unique partners

• Cell cycle-specific analysis:

  • Synchronize cells and analyze ZC3HC1:

    • Localization (nuclear basket versus nucleoplasmic/cytoplasmic)

    • Ubiquitination activity across cell cycle

    • Post-translational modifications

  • Earlier research described ZC3HC1/NIPA as "targeting cyclin B1 (CCNB1) in interphase, to promote its degradation"

• In vitro reconstitution:

  • Purify components of:

    • Nuclear basket (TPR, ZC3HC1)

    • SCF complex (SKP1, CUL1, ZC3HC1)

  • Test if basket-bound ZC3HC1 can still participate in SCF complex formation

  • Assess ubiquitination activity of nuclear basket-bound versus soluble ZC3HC1

• Proteomic analysis of substrates:

  • Identify ubiquitination substrates of ZC3HC1 when:

    • Bound to nuclear basket

    • In soluble nucleoplasmic/cytoplasmic pools

  • Determine if substrate profiles differ based on localization

These methodologies would provide mechanistic insights into the relationship between ZC3HC1's dual roles and determine whether they represent independent functions or coordinated activities that integrate nuclear envelope dynamics with cell cycle control.

How can ZC3HC1 antibodies be utilized to investigate its role in coronary artery disease and cancer progression?

ZC3HC1 has been associated with both coronary artery disease through SNPs and altered expression in cancers . These methodological approaches can investigate these relationships:

• Genetic variant-specific analysis:

  • Develop assays to distinguish between ZC3HC1 variant proteins

  • Generate cell lines carrying disease-associated SNPs using CRISPR-Cas9

  • Compare:

    • Protein stability and half-life

    • Nuclear basket localization efficiency

    • Interaction with TPR and other partners

    • Cell cycle regulatory functions

• Tissue microarray analysis:

  • Perform immunohistochemistry on cardiovascular disease and cancer tissue microarrays

  • Quantify:

    • ZC3HC1 expression levels

    • Subcellular localization patterns

    • Correlation with disease progression markers

  • Compare with genetic information when available

• Multi-parameter flow cytometry:

  • Develop protocols for simultaneous detection of:

    • ZC3HC1 expression levels

    • Cell cycle status

    • Disease-specific markers

  • Apply to patient-derived samples

  • Correlate with clinical outcomes

• Functional models of disease-associated variants:

  • Express disease-associated ZC3HC1 variants in appropriate cell types:

    • Vascular endothelial cells for coronary artery disease

    • Cancer cell lines for tumor progression studies

  • Assess:

    • Nuclear envelope structure and function

    • Cell cycle progression patterns

    • Transcriptional responses

    • Cellular migration and invasion (for cancer models)

• Integrative multi-omics:

  • Combine:

    • ZC3HC1 immunoprofiling

    • Transcriptomics

    • Chromatin accessibility

    • Proteomics

  • Identify disease-specific changes in nuclear basket function and downstream effects

These approaches provide mechanistic insights into how ZC3HC1 variants or expression changes contribute to disease pathogenesis and potential therapeutic strategies.

What are the methodological considerations for studying ZC3HC1 phosphorylation states across different cellular conditions?

Research indicates ZC3HC1 undergoes phosphorylation , which may regulate its function. To study these modifications:

• Phosphorylation-specific detection methods:

  • Phospho-specific antibody development:

    • Generate antibodies against predicted phosphosites

    • Validate specificity using phosphatase treatments

  • Phos-tag™ gel electrophoresis:

    • Separate phosphorylated ZC3HC1 isoforms

    • Quantify relative abundance across conditions

• Mass spectrometry-based approaches:

  • Enrichment strategies:

    • Immunoprecipitate ZC3HC1 from different cellular states

    • Enrich phosphopeptides using TiO₂ or IMAC

  • Targeted approaches:

    • Parallel reaction monitoring (PRM)

    • Multiple reaction monitoring (MRM)

  • Quantitative comparison across conditions:

    • SILAC or TMT labeling for relative quantification

    • Absolute quantification using synthetic phosphopeptide standards

• Phosphorylation dynamics across cell cycle:

  • Synchronize cells at different stages

  • Research by Gunkel et al. used "Lambda protein phosphatase (NEB)" in experiments to study ZC3HC1 phosphorylation

  • Quantify changes in phosphorylation status

  • Correlate with functional transitions:

    • Nuclear basket association

    • SCF complex formation

    • Cell cycle regulation

• Kinase-substrate relationship identification:

  • In vitro kinase assays with candidate kinases

  • Kinase inhibitor screens to identify relevant pathways

  • Phosphosite mutant analysis (Ala/Glu substitutions)

  • Correlation with known cell cycle kinase activities

• Visualization of phosphorylation in situ:

  • Proximity ligation assays using:

    • Anti-ZC3HC1 antibodies

    • Anti-phospho-epitope antibodies

  • Live-cell imaging with phosphorylation-sensitive biosensors

These methodological approaches provide comprehensive insights into ZC3HC1 phosphorylation dynamics and their functional significance in both normal cellular processes and disease states.

How can new genomic and proteomic technologies be applied to advance ZC3HC1 research?

Cutting-edge technologies offer new opportunities for ZC3HC1 research:

• Spatial proteomics approaches:

  • Proximity labeling (BioID, APEX) fused to ZC3HC1

  • Quantitative mapping of the nuclear basket interactome

  • Comparison across cell types and conditions

  • Research on ZC3HC1's NuBaID provides foundation for targeted interactome mapping

• CRISPR-based functional genomics:

  • Genome-wide screens for modifiers of ZC3HC1 function

  • CRISPRa/CRISPRi to modulate ZC3HC1 expression

  • Base editing to introduce disease-associated variants

  • Perturb-seq to link genetic perturbations to transcriptional responses

• Quantitative image analysis:

  • High-content screening of ZC3HC1 localization

  • Single-molecule tracking of ZC3HC1 dynamics

  • Correlative light-electron microscopy of nuclear basket structure

  • Machine learning-based analysis of nuclear envelope morphology

• Structural biology advances:

  • AlphaFold2 predictions of ZC3HC1 structure have already provided insights

  • Cryo-electron tomography of the nuclear basket

  • In-cell NMR to study ZC3HC1 conformational states

  • Integrative structural modeling of the nuclear basket complex

• Single-cell multi-omics:

  • Correlate ZC3HC1 protein levels with transcriptome

  • Map cell cycle-dependent changes

  • Identify cell state-specific functions

  • Link to chromatin organization at the nuclear periphery

These technologies enable systematic investigation of ZC3HC1 biology at unprecedented resolution and scale, potentially revealing new functions and regulatory mechanisms.

What are the emerging hypotheses about the evolutionary significance of ZC3HC1's dual roles in nuclear structure and cell cycle regulation?

Recent discoveries about ZC3HC1's evolutionary conservation and dual functionality raise intriguing hypotheses:

• Coordinated evolution of nuclear architecture and cell cycle:

  • Research indicates "species with a ZC3HC1 homologue also have a TPR/Mlp homologue," suggesting co-evolution

  • Hypothesis: ZC3HC1 may represent an evolutionary link that coordinates nuclear envelope remodeling with cell cycle progression

  • Testing approach: Comparative analysis of ZC3HC1-TPR interactions across diverse eukaryotic lineages

• Nuclear basket as a cell cycle checkpoint platform:

  • ZC3HC1's dual role may position the nuclear basket as a regulatory hub

  • Hypothesis: Nuclear basket may integrate nuclear transport signals with cell cycle status

  • Testing approach: Analyze nuclear transport efficiency in cells with mutations in ZC3HC1's separate functional domains

• Compartmentalization of ubiquitination activities:

  • The nuclear basket-bound ZC3HC1 may target different substrates than its soluble form

  • Hypothesis: Spatial organization of E3 ligase activity provides regulatory specificity

  • Testing approach: Proximity labeling of ZC3HC1 substrates when tethered to different cellular compartments

• Evolutionary repurposing of structural components:

  • Research revealed that "ZC3HC1 and its homologues stand out as unique"

  • Hypothesis: ZC3HC1 represents an example of evolutionary co-option where structural proteins acquired regulatory functions

  • Testing approach: Functional analysis of ZC3HC1 homologs from diverse lineages to identify ancestral functions

• Integration of mechanosensing and cell cycle control:

  • Nuclear envelope mechanics could influence cell cycle decisions

  • Hypothesis: ZC3HC1 may transduce mechanical signals from the nuclear envelope to cell cycle machinery

  • Testing approach: Measure ZC3HC1-dependent responses to altered nuclear mechanics

These hypotheses represent cutting-edge directions in understanding the evolutionary and functional significance of ZC3HC1's dual roles, with implications for fundamental cell biology and disease mechanisms.