NUP214 Antibody

What is NUP214 and what cellular functions does it perform?

NUP214, also known as CAN, is a 214 kDa nuclear pore complex (NPC) protein containing multiple FG-peptide sequence motifs. It serves as a critical component anchored to the cytoplasmic ring of the nuclear pore complex. NUP214 forms a subcomplex with the nucleoporin NUP88 and interacts with hCRM1 (exportin 1), a nuclear export receptor. The protein plays an essential role in nucleocytoplasmic transport, specifically in protein and mRNA nuclear export processes. Expression studies have shown that NUP214 is predominantly found in thymus, spleen, bone marrow, kidney, brain, and testis, but is minimally expressed in other tissues or during embryonic development . Research has demonstrated that depletion of NUP214 in knockout mouse embryonic cells results in cell cycle arrest in G2 phase, followed by inhibition of nuclear protein import and blocked mRNA export, underscoring its critical importance in cellular functions .

What are the validated applications for NUP214 antibodies in research?

NUP214 antibodies have been validated for multiple experimental applications, providing researchers with versatile tools for studying this nucleoporin. The primary validated applications include:

When using these applications, researchers should note that antigen retrieval may be necessary for IHC applications, with suggested protocols including TE buffer (pH 9.0) or citrate buffer (pH 6.0) . Additionally, it is recommended that researchers optimize dilutions for each specific testing system to obtain optimal results, as signal strength can be sample-dependent .

How should researchers prepare samples for optimal NUP214 antibody detection?

For optimal detection of NUP214 using antibodies, researchers should follow application-specific preparation protocols. For immunocytochemistry applications, cell samples should be fixed, permeabilized, and immunostained according to established protocols. This typically involves fixation with paraformaldehyde, permeabilization with a detergent such as Triton X-100, and blocking with appropriate blocking solutions to minimize non-specific binding. Primary antibodies should be diluted according to manufacturer recommendations - for example, anti-C-terminal CAN (CNC) antibodies are typically diluted 1:400, while other antibodies may require different dilutions .

For Western blot applications, proper sample preparation is crucial. Cells should be lysed in appropriate buffers containing protease inhibitors, and proteins should be denatured in sample buffer containing SDS and a reducing agent. For NUP214 detection specifically, the observed molecular weight is typically around 250 kDa, which is consistent with literature descriptions . This higher observed weight compared to the calculated molecular weight (213 kDa based on 2080 amino acids) should be taken into consideration when evaluating Western blot results.

What are the recommended storage conditions for NUP214 antibodies?

Proper storage of NUP214 antibodies is essential for maintaining their activity and specificity. Based on manufacturer recommendations, most NUP214 antibodies should be stored at -20°C, where they typically remain stable for one year after shipment. The antibodies are generally provided in a storage buffer containing PBS with 0.02% sodium azide and 50% glycerol at pH 7.3 . Importantly, aliquoting is often unnecessary for -20°C storage, which can help prevent freeze-thaw cycles that might degrade antibody quality. Some antibody preparations may contain small amounts (0.1%) of BSA as a stabilizer. Researchers should always refer to specific product information, as storage requirements may vary slightly between manufacturers and antibody formulations .

How does NUP214 relate to the Notch signaling pathway, and what methodologies can detect this interaction?

NUP214 has been identified as a negative regulator of Notch signaling through its role in nuclear trafficking. Research has demonstrated that knockdown of NUP214 enhances Notch signaling activity, as measured by CSL-luciferase reporter assays. This relationship appears to be mediated through the nuclear trafficking of RBP-J (CSL), an essential component of canonical Notch signaling that forms a co-activator complex with NICD to drive Notch-dependent transcription .

For researchers investigating this relationship, the following methodological approaches are recommended:

• CSL-luciferase reporter assays: Knockdown of NUP214 in PC3 cells increases luciferase expression from a 12× CSL-luciferase reporter plasmid. This assay can be performed by transfecting cells with siRNA targeting NUP214 for 48 hours, followed by transfection with the reporter plasmid and a Renilla luciferase control plasmid. Luciferase activities should be measured 24 hours after plasmid transfection using standard dual-luciferase protocols .

• Differentiation assays in C2C12 cells: C2C12 myoblasts normally differentiate into myotubes upon serum withdrawal, a process that is inhibited by Notch signaling. Knockdown of NUP214 has been shown to delay or reduce differentiation of these cells, which can be measured by the ratio of myosin heavy chain (MyHC) positive cells to total cells, the fusion index, and MyHC protein levels. This effect can be rescued by additional knockdown of Hes1, a major Notch downstream gene in myogenesis, confirming that the differentiation block by NUP214 knockdown is mediated via Notch signaling .

• Chromatin immunoprecipitation (ChIP): ChIP-qPCR using validated RBP-J antibodies can detect increased binding of RBP-J to Notch target genes following NUP214 knockdown. This technique provides direct evidence of NUP214's role in regulating the DNA-binding activity of RBP-J at Notch target genes .

These methodologies provide complementary approaches to investigate the functional relationship between NUP214 and Notch signaling in different cellular contexts.

What are the consequences of NUP214 overexpression in experimental systems?

Overexpression of NUP214/CAN in experimental systems has revealed significant cellular consequences, providing insights into its normal function and potential pathological roles. In U937 myeloid precursor cells, overexpression of CAN resulted in cell cycle arrest in G0, accumulation of mRNA in the nucleus, and eventual apoptotic cell death. These effects contrast with the overexpression of the leukemia-associated fusion protein DEK-CAN, which did not interfere with terminal myeloid differentiation of these cells .

The mechanisms underlying these effects involve the mislocalization of nuclear transport factors. Immunofluorescence studies have demonstrated that ectopically expressed CAN protein causes colocalization of hCRM1 (exportin 1) and import factor p97/importin β, effectively depleting these factors from the nuclear pore complex . This disruption of the nuclear transport machinery highlights the importance of maintaining correct stoichiometry of nuclear pore complex components.

For researchers studying NUP214 overexpression, the following methodological considerations are important:

• Use inducible expression systems (such as tetracycline-repressible systems) to control expression levels and timing

• Include appropriate controls for cell viability and proliferation, as NUP214 overexpression can inhibit cell growth

• Monitor subcellular localization of transport factors using immunofluorescence

• Assess mRNA export using appropriate RNA visualization techniques

• Measure cell cycle distribution and apoptotic markers to characterize cellular responses

These findings also suggest that researchers should be cautious when interpreting results from overexpression studies, as the cellular effects may reflect disruption of nuclear transport machinery rather than the normal function of NUP214.

What protocols are recommended for studying NUP214's role in nucleocytoplasmic transport?

To effectively study NUP214's role in nucleocytoplasmic transport, researchers should employ multiple complementary approaches:

Nucleocytoplasmic transport assays: Nuclear export can be assessed using fluorescent reporter proteins fused to nuclear export signals (NES). For example, activation-induced deaminase (AID)-GFP serves as a known CRM1 substrate for studying nuclear export pathways. Researchers can transfect cells with the reporter construct, then manipulate NUP214 expression through knockdown or overexpression, and monitor the subcellular distribution of the reporter by fluorescence microscopy. Treatment with Leptomycin B (LMB), a specific inhibitor of CRM1-dependent export, serves as a positive control .

Subcellular fractionation: For biochemical assessment of protein distribution between nuclear and cytoplasmic compartments, subcellular fractionation followed by Western blotting can be employed. This approach allows quantification of the nuclear/cytoplasmic ratio of proteins of interest, such as transport factors or cargo proteins, under conditions of NUP214 manipulation.

Immunofluorescence detection of endogenous transport factors: Localization of transport machinery components can be visualized by immunofluorescence microscopy. For example, the distribution of RBP-J can be quantified following NUP214
knockdown, revealing a significant shift toward nuclear accumulation . Primary antibodies should be carefully validated, and appropriate controls should be included to ensure specificity.

RNA export assessment: Since NUP214 plays a role in mRNA export, researchers can visualize nuclear RNA accumulation using fluorescence in situ hybridization (FISH) with oligo(dT) probes to detect poly(A)+ RNA. This approach can reveal export defects associated with NUP214 dysfunction.

How can researchers optimize Western blot protocols for detecting NUP214?

Detection of NUP214 by Western blot requires optimization due to its high molecular weight (observed at approximately 250 kDa) and potentially variable expression levels across different cell types. Researchers should implement the following protocol optimizations:

• Sample preparation: Use lysis buffers containing proper protease inhibitors to prevent degradation. Include phosphatase inhibitors if studying phosphorylation states of NUP214.

• Gel electrophoresis: Employ low percentage (6-8%) SDS-PAGE gels or gradient gels that allow better resolution of high molecular weight proteins. Extended running times may be necessary for proper separation.

• Transfer conditions: Use wet transfer methods with reduced methanol concentration in transfer buffer (5-10% instead of the standard 20%) to facilitate transfer of high molecular weight proteins. Consider extending transfer time or using specialized transfer systems designed for large proteins.

• Blocking and antibody incubation:

  • Recommended primary antibody dilution: 1:1000-1:6000 for Western blot applications

  • Optimal blocking conditions typically involve 5% non-fat dry milk or BSA in TBST

  • Extended primary antibody incubation (overnight at 4°C) often yields better results

• Detection system: Use high-sensitivity detection reagents, particularly for cell types with lower expression levels of NUP214.

• Positive controls: Include K-562 cells as a positive control, as they have been validated for NUP214 detection .

Researchers should note that the observed molecular weight of NUP214 (250 kDa) is higher than the calculated molecular weight based on amino acid sequence (213 kDa), which is consistent with findings in the literature . This discrepancy should be considered when evaluating Western blot results.

What are the critical considerations when using NUP214 antibodies in studying leukemia-associated NUP214 fusions?

NUP214 is involved in chromosomal translocations associated with leukemogenesis, making antibodies against this protein valuable tools in leukemia research . When studying leukemia-associated NUP214 fusions such as DEK-CAN, researchers should consider several critical factors:

• Epitope mapping: Determine whether the antibody recognizes an epitope retained in the fusion protein. NUP214 antibodies generated against C-terminal epitopes may not detect N-terminal fusion proteins like DEK-CAN. Conversely, antibodies against the N-terminus of NUP214 would be required to detect fusion proteins where the C-terminus is lost.

• Differential localization: While wild-type NUP214 localizes to the nuclear pore complex, fusion proteins often show altered subcellular localization. Immunofluorescence protocols should be optimized to detect these distribution differences. Use confocal laser-scanning microscopy with appropriate controls to accurately assess localization patterns .

• Expression systems: When studying fusion proteins, consider using inducible expression systems to control expression levels and timing. This approach is particularly important since overexpression of both wild-type NUP214 and fusion proteins can be cytotoxic .

• Functional assays: Beyond detection, assess the functional impact of NUP214 fusions using appropriate assays:

  • For DEK-CAN, evaluate effects on myeloid differentiation in relevant cell lines such as U937

  • Monitor nucleocytoplasmic transport using reporter systems

  • Assess impacts on cell cycle progression and proliferation

• Control experiments: Include appropriate controls such as wild-type NUP214 expression and empty vector controls when conducting functional studies with fusion proteins.

By carefully considering these factors, researchers can obtain more reliable and interpretable results when studying leukemia-associated NUP214 fusions using antibody-based approaches.

How can researchers validate the specificity of NUP214 antibodies in their experimental systems?

Validating antibody specificity is crucial for obtaining reliable research results. For NUP214 antibodies, researchers should employ multiple complementary approaches:

• Knockdown/knockout validation: Perform siRNA knockdown or CRISPR/Cas9 knockout of NUP214 and verify reduction or loss of signal by Western blot and immunostaining. For siRNA approaches, researchers can use protocols similar to those described in the literature, transfecting cells with 10 nM siRNA using appropriate transfection reagents such as Dharmafect .

• Overexpression validation: Express tagged versions of NUP214 and confirm co-localization with antibody staining or co-migration on Western blots.

• Peptide competition: Pre-incubate the antibody with the immunizing peptide (if available) before application to samples. Specific signals should be blocked by this treatment.

• Multiple antibodies comparison: Use different antibodies targeting distinct epitopes of NUP214 and compare their staining patterns.

• Cross-species reactivity assessment: Test the antibody in samples from different species to confirm expected patterns of reactivity. Available data indicates that certain NUP214 antibodies show reactivity with human and mouse samples, with cited reactivity for human, rat, and pig samples .

• Positive control tissues/cells: Include known positive controls in experiments. For NUP214, K-562 cells serve as a reliable positive control for Western blot, while mouse kidney tissue provides a good positive control for immunohistochemistry applications .

These validation approaches should be documented and reported in publications to enhance reproducibility and reliability of research findings.

What are common pitfalls in immunohistochemistry applications of NUP214 antibodies and how can they be addressed?

Immunohistochemistry (IHC) with NUP214 antibodies presents several challenges that researchers should anticipate and address:

• Antigen retrieval issues: NUP214 detection in tissues often requires specific antigen retrieval conditions. For optimal results, it is recommended to use TE buffer at pH 9.0 for antigen retrieval. Alternatively, citrate buffer at pH 6.0 can be used, though it may yield different results . Researchers should compare both methods to determine which works best for their specific tissue and fixation conditions.

• Fixation artifacts: Overfixation can mask epitopes and reduce antibody binding. Standardize fixation protocols (duration and fixative concentration) for consistent results.

• Background staining: High background can obscure specific signals. This can be addressed by:

  • Using optimal antibody dilutions (1:250-1:1000 range is recommended for IHC applications)

  • Extending blocking steps with appropriate blocking reagents

  • Including additional washing steps with detergent-containing buffers

  • Using more specific detection systems

• Tissue-specific expression variations: NUP214 is expressed at different levels across tissues, with higher expression in thymus, spleen, bone marrow, kidney, brain, and testis, but limited expression in other tissues . This variation should be considered when interpreting negative results in certain tissues.

• Control inclusion: Always include positive control tissues (such as mouse kidney tissue) and negative controls (primary antibody omitted) in each IHC run to validate staining specificity.

• Counterstaining optimization: Adjust counterstaining intensity to avoid masking specific NUP214 signals while maintaining sufficient context for tissue architecture visualization.

By addressing these potential pitfalls, researchers can obtain more reliable and interpretable IHC results with NUP214 antibodies.

How do experimental conditions impact the performance of NUP214 antibodies in different assays?

Various experimental conditions can significantly impact the performance of NUP214 antibodies across different applications. Understanding these factors is essential for optimizing experimental protocols:

• Temperature effects:

  • For Western blot applications, primary antibody incubation can be performed at 4°C overnight or at room temperature for 1-2 hours, with the former often providing better signal-to-noise ratios

  • For immunofluorescence, room temperature incubations are typically sufficient, but temperature stability during incubation periods is important for consistent results

• Buffer composition impacts:

  • The storage buffer (PBS with 0.02% sodium azide and 50% glycerol at pH 7.3) helps maintain antibody stability, but must be sufficiently diluted in working solutions

  • Blocking buffers containing BSA versus non-fat dry milk may yield different results depending on the specific antibody and sample type

  • Addition of detergents (like Tween-20 or Triton X-100) at appropriate concentrations can reduce non-specific binding while maintaining specific signals

• Incubation time considerations:

  • Extended primary antibody incubation times generally improve signal intensity but may increase background

  • Secondary antibody incubation times should be carefully optimized, as longer incubations don't necessarily improve results and may increase non-specific binding

• Fixation method effects:

  • For immunofluorescence and IHC, the choice between paraformaldehyde, methanol, or other fixatives can dramatically affect epitope accessibility

  • Fixation duration is equally important, with overfixation potentially masking epitopes

• Detection system sensitivity:

  • Enzymatic detection systems (like HRP) with amplification steps may provide higher sensitivity for low-abundance targets

  • Fluorescent detection systems offer better spatial resolution and quantification capabilities

By systematically optimizing these parameters for specific experimental contexts, researchers can significantly improve the performance and reliability of NUP214 antibody-based assays.

How can NUP214 antibodies be used to study its role in leukemogenesis?

NUP214 has been implicated in leukemogenesis through chromosomal translocations, making it an important target for cancer research. Researchers can employ several approaches using NUP214 antibodies to investigate its role in leukemic processes:

• Detection of fusion proteins: NUP214 antibodies can be used to detect leukemia-associated fusion proteins like DEK-CAN, provided the antibody epitope is retained in the fusion. Researchers should select antibodies targeting the appropriate region of NUP214 and validate them in systems expressing the fusion proteins of interest .

• Expression profiling in patient samples: Immunohistochemistry using NUP214 antibodies can reveal expression patterns in bone marrow biopsies from leukemia patients. This approach allows comparison of NUP214 expression levels between normal and leukemic cells, potentially identifying aberrant expression patterns associated with disease progression.

• Functional studies in leukemia models: By combining NUP214 antibodies with functional assays, researchers can investigate the consequences of NUP214 alterations:

  • Use immunofluorescence to track subcellular localization changes in leukemic cells

  • Employ co-immunoprecipitation to identify altered protein interactions in leukemia contexts

  • Apply ChIP techniques to study potential transcriptional regulatory roles

• Therapeutic target validation: As NUP214 may represent a potential therapeutic target, antibodies can be used to validate target engagement in drug development pipelines. This includes monitoring changes in NUP214 expression, localization, or interactions following treatment with candidate therapeutics.

These approaches provide complementary insights into the complex roles of NUP214 in leukemogenesis, potentially revealing new diagnostic markers or therapeutic targets.

What are the protocols for studying NUP214's interaction with the Notch signaling pathway in disease contexts?

The interaction between NUP214 and the Notch signaling pathway has important implications for disease processes, particularly in contexts where aberrant signaling contributes to pathology. Researchers can employ the following methodological approaches to investigate this relationship:

• Luciferase reporter assays: To assess Notch pathway activity in response to NUP214 manipulation, researchers can use CSL-luciferase reporter assays following specific protocols:

  • Transfect cells with siRNA targeting NUP214 (10 nM concentration using appropriate transfection reagents)

  • After 48 hours, transfect cells with the pGL4.20–12× CSL-luciferase reporter plasmid (300 ng) and a Renilla luciferase control plasmid (6 ng)

  • Measure luciferase activities 24 hours after plasmid transfection using standard dual-luciferase protocols

• ChIP-qPCR for RBP-J binding: This technique directly assesses how NUP214 affects RBP-J binding to Notch target genes:

  • Perform chromatin immunoprecipitation with validated RBP-J antibodies following NUP214 knockdown

  • Use qPCR primers targeting known RBP-J binding sites in Notch target genes

  • Include control regions not bound by RBP-J to demonstrate specificity

• Co-immunoprecipitation studies: To investigate physical interactions between NUP214 and Notch pathway components:

  • Use anti-NUP214 antibodies for immunoprecipitation from cell lysates

  • Probe Western blots for Notch pathway components to detect potential interactions

  • Include appropriate controls (IgG control immunoprecipitations, input samples)

• Cell differentiation assays: The C2C12 myoblast differentiation model provides a functional readout of Notch pathway activity:

  • Knockdown NUP214 in C2C12 cells

  • Induce differentiation by serum withdrawal

  • Assess differentiation by measuring myosin heavy chain (MyHC) positive cells and calculating the fusion index

  • Include Hes1 knockdown as a rescue condition to confirm Notch pathway involvement

These methodological approaches provide complementary insights into how NUP214 regulates Notch signaling in various disease contexts.

How can NUP214 antibodies be incorporated into multi-omics research approaches?

As multi-omics approaches become increasingly important in biological research, NUP214 antibodies can be integrated into these comprehensive studies through several methodologies:

• Proteomics integration: NUP214 antibodies can be used for immunoprecipitation followed by mass spectrometry (IP-MS) to identify novel interaction partners and post-translational modifications. This approach can reveal how NUP214's interactome changes under different cellular conditions or in disease states. For example, research has identified interactions between NUP214 and transport factors such as hCRM1, providing insights into nucleocytoplasmic transport mechanisms .

• Chromatin immunoprecipitation sequencing (ChIP-seq): Although NUP214 is primarily associated with the nuclear pore complex, research has shown that nucleoporins can have roles in gene regulation. ChIP-seq using NUP214 antibodies can identify potential genomic binding sites and, when combined with RNA-seq, reveal correlations between NUP214 genome associations and gene expression patterns.

• Spatial transcriptomics combinations: Combining immunofluorescence using NUP214 antibodies with in situ RNA detection techniques can reveal spatial relationships between NUP214 localization and specific mRNA export or localization patterns. This is particularly relevant given NUP214's role in mRNA export.

• Functional genomics integration: NUP214 antibodies can validate the effects of genetic perturbations (CRISPR screens, siRNA) on protein expression and localization, connecting genomic alterations to proteomic consequences.

• Single-cell approaches: Integration of NUP214 immunostaining with single-cell RNA-seq or proteomics can reveal cell-to-cell variability in NUP214 expression and its correlation with transcriptional or proteomic profiles.

These integrated approaches leverage the specificity of NUP214 antibodies within broader multi-omics frameworks to provide more comprehensive insights into biological systems and disease mechanisms.

What are the emerging applications of NUP214 antibodies in studying nuclear transport defects in neurodegenerative diseases?

While historically NUP214 research has focused on its role in leukemia, emerging evidence suggests potential roles for nuclear transport defects in neurodegenerative diseases. Researchers can apply NUP214 antibodies to investigate these connections through the following approaches:

• Comparative expression analysis: Using immunohistochemistry with NUP214 antibodies to compare expression patterns in normal versus diseased brain tissue may reveal alterations associated with neurodegenerative conditions. The documented expression of NUP214 in brain tissue makes this a feasible approach .

• Co-localization studies: Combining NUP214 immunofluorescence with markers of neurodegenerative disease-associated proteins (such as tau, amyloid-β, or TDP-43) can reveal potential spatial relationships between nuclear pore components and disease-specific protein aggregates.

• Nuclear transport functional assays: Using reporter constructs to assess nuclear transport efficiency in neuronal models, coupled with NUP214 immunostaining, can link structural changes in the nuclear pore complex with functional transport defects in disease contexts.

• Post-translational modification analysis: Immunoprecipitation with NUP214 antibodies followed by mass spectrometry can identify disease-specific post-translational modifications that might affect nuclear pore function in neurodegenerative conditions.

• Age-related changes assessment: Given that both nuclear transport defects and neurodegenerative diseases increase with age, systematic immunohistochemical analysis of NUP214 expression and localization across age groups may reveal correlations relevant to disease progression.

These approaches represent emerging directions for applying NUP214 antibodies beyond their traditional research contexts, potentially contributing to our understanding of nuclear transport defects in neurodegeneration.