NUP98A Antibody

What is NUP98 and what cellular functions does it perform?

NUP98 (Nucleoporin 98kDa) is a component of the nuclear pore complex (NPC), which are large protein assemblies embedded in the nuclear envelope. It plays a critical role in nucleocytoplasmic trafficking, interacting with several transport factors to regulate the movement of molecules between the nucleus and cytoplasm . Beyond its structural role in nuclear transport, NUP98 has been identified as a potential tumor suppressor that regulates select p53 target genes, including p21 . This function is distinct from the well-characterized oncogenic properties of NUP98 fusion proteins frequently observed in leukemias . NUP98 has a molecular weight of approximately 98 kDa, though the observed molecular weight in experimental conditions is often around 105 kDa .

How does NUP98 regulate gene expression and cellular processes?

NUP98 regulates gene expression through multiple mechanisms. Most notably, it stabilizes mature mRNA of specific targets, particularly p21. Studies have demonstrated that NUP98 interacts with the 3'-UTR region of p21 mRNA, extending its half-life from approximately 45 minutes to 90 minutes . This post-transcriptional regulation is independent of transcription initiation and splicing processes. NUP98's regulatory function appears to be selective, as it does not similarly affect all p53 targets - for example, PUMA expression remains unaltered by NUP98 knockdown . Through these mechanisms, NUP98 contributes to important cellular processes including cell cycle regulation, DNA damage response, and cellular senescence .

What are the key considerations when selecting a NUP98 antibody for research?

When selecting a NUP98 antibody, researchers should consider:

• Specificity: Confirm the antibody specifically recognizes NUP98, as evidenced by immunoblotting validation. For example, MAb 2H10 has been demonstrated to specifically recognize human NUP98 .

• Applications compatibility: Ensure the antibody is validated for your intended application (WB, IHC, IF/ICC, IP). For instance, the NUP98-NUP96 antibody (12329-1-AP) is validated for multiple applications including Western blot (1:500-1:4000 dilution), immunoprecipitation (0.5-4.0 μg for 1.0-3.0 mg total protein), immunohistochemistry (1:50-1:500), and immunofluorescence (1:50-1:500) .

• Species reactivity: Verify reactivity with your experimental model. Some antibodies, like 12329-1-AP, have been tested in human samples but also cited for reactivity with mouse and pig samples .

• Epitope recognition: Consider whether the antibody recognizes specific regions or isoforms of NUP98, particularly if studying NUP98 fusion proteins or specific domains.

• Technical considerations: Review recommended storage conditions, buffer compatibility, and optimal protocols for antigen retrieval when applicable .

What are the optimal conditions for using NUP98 antibodies in Western blotting?

For optimal Western blotting with NUP98 antibodies:

• Sample preparation: Based on validated cell lines, prepare lysates from appropriate cells (e.g., COLO 320, Jurkat, HeLa, K-562, or MCF-7 cells) .

• Loading and separation: Load sufficient protein (typically 20-50 μg) and ensure adequate separation of higher molecular weight proteins (>100 kDa) by using appropriate percentage acrylamide gels (typically 6-8%).

• Transfer conditions: Use wet transfer methods with standard PVDF or nitrocellulose membranes. Extended transfer times (1-2 hours) may improve transfer efficiency for this high molecular weight protein.

• Antibody dilution: For antibody 12329-1-AP, use dilutions between 1:500-1:4000. The exact dilution should be optimized for each experimental system .

• Detection: When using NUP98 antibodies, particularly note that the observed molecular weight is often approximately 105 kDa, slightly higher than the calculated 98 kDa .

• Controls: Include positive controls from validated cell lines and consider using siRNA knockdown samples as negative controls to confirm specificity, as demonstrated in previous studies .

How can immunoprecipitation with NUP98 antibodies be optimized for studying protein-protein and protein-RNA interactions?

Optimizing immunoprecipitation with NUP98 antibodies for interaction studies:

• Antibody selection: Choose antibodies validated for IP applications, such as 12329-1-AP, which requires 0.5-4.0 μg antibody for 1.0-3.0 mg of total protein lysate .

• Cross-linking considerations: For transient interactions, consider using reversible cross-linking agents to stabilize complexes.

• Protein-protein interactions: For studying NUP98 interactions with other proteins (e.g., TPR, Nup96), use gentle lysis conditions that preserve protein complexes. Research has demonstrated that Nup98 interacts with TPR (a component of intranuclear filamentous structures) and that Nup96 interacts indirectly with TPR via Nup98 .

• RNA immunoprecipitation: For studying NUP98-RNA interactions (particularly important for p21 mRNA stabilization):

  • Include RNase inhibitors in all buffers

  • Consider using formaldehyde cross-linking to preserve RNA-protein interactions

  • Use appropriate controls including IgG control and input RNA

  • Design primers for specific regions (e.g., 3'-UTR of p21 mRNA) as NUP98 shows strongest interaction with the 3'-UTR region

• Verification: Confirm successful immunoprecipitation by Western blotting a small aliquot of the IP sample before proceeding to downstream applications.

What are the recommended protocols for immunofluorescence staining of NUP98 in different cell types?

Recommended immunofluorescence protocols for NUP98 staining:

• Fixation and permeabilization:

  • For standard fixation: 4% paraformaldehyde (10-15 minutes) followed by 0.2% Triton X-100 permeabilization

  • Alternative approach: Methanol fixation (-20°C, 10 minutes) which simultaneously fixes and permeabilizes

• Blocking: 5% normal serum (matched to secondary antibody host) with 0.3% Triton X-100 for 1 hour at room temperature

• Primary antibody: For NUP98-NUP96 antibody (12329-1-AP), use 1:50-1:500 dilution ; incubate overnight at 4°C

• Expected staining pattern: A punctate nuclear rim staining pattern is typically observed with NUP98 antibodies . When studying the intranuclear distribution, specific structures including perinucleolar rings and filaments extending to the nuclear periphery may be visible .

• Co-localization studies: For co-localization with other nuclear pore components or interacting proteins (e.g., TPR), double immunofluorescence can be performed. Previous studies have shown co-localization of TPR and Nup98 in the nuclear interior .

• Technical considerations:

  • Use confocal microscopy for optimal resolution of nuclear pore structures

  • Consider using z-stack imaging to fully capture the three-dimensional distribution of NUP98

How can NUP98 antibodies be used to investigate its role in tumor suppression and cancer biology?

NUP98 antibodies can be employed in multiple approaches to investigate its tumor suppressive functions:

• Expression analysis in cancer tissues:

  • Use immunohistochemistry with NUP98 antibodies on tissue microarrays to assess expression levels across tumor samples

  • Previous research has shown reduced expression of Nup98 in murine and human hepatocellular carcinomas (HCC), correlating with p21 expression in HCC patients

  • For IHC applications with antibody 12329-1-AP, use dilutions of 1:50-1:500 with appropriate antigen retrieval (TE buffer pH 9.0 or citrate buffer pH 6.0)

• Mechanistic studies:

  • Combine NUP98 antibodies with RNA immunoprecipitation to identify additional mRNA targets beyond p21

  • An in silico approach revealed 14-3-3σ as another p53 target regulated by Nup98, suggesting broader regulatory functions

• Functional assays:

  • Use NUP98 antibodies to monitor protein levels after siRNA knockdown in senescence and apoptosis assays

  • Previous studies demonstrated that Nup98 knockdown diminished senescence-associated beta-galactosidase activity (from 43±15% to 12±9% positive cells) following Nutlin treatment

• Protein complex analysis:

  • Investigate how NUP98 interacts with p53 pathway components using co-immunoprecipitation with NUP98 antibodies

  • Explore potential changes in these interactions across normal and cancer cells

What approaches can be used to study NUP98 fusion proteins in leukemia research?

For studying NUP98 fusion proteins in leukemia research:

• Antibody selection considerations:

  • Determine whether the antibody epitope is retained in fusion proteins

  • Consider using antibodies targeting the N-terminal region of NUP98, which is typically preserved in fusion proteins like NUP98-KDM5A

• Detection of fusion proteins:

  • Western blotting: Optimize gel percentage to resolve higher molecular weight fusion proteins

  • Immunofluorescence: Compare localization patterns between wild-type NUP98 and fusion proteins, as fusion proteins often show altered nuclear distribution

• Model systems:

  • Human pluripotent stem cell (hPSC) models with inducible expression of NUP98 fusion proteins (e.g., NUP98-KDM5A) allow controlled timing of oncogene expression during hematopoietic development

  • Use immunoblotting with NUP98 antibodies to confirm fusion protein expression in these systems

• Mechanistic investigations:

  • Chromatin immunoprecipitation (ChIP) using NUP98 antibodies to identify genomic binding sites of fusion proteins

  • RNA-seq following immunoprecipitation to compare mRNA targets of wild-type NUP98 versus fusion proteins

  • Assess effects on p21 and other target mRNAs, as the fusion proteins may lack the regulatory functions of wild-type NUP98

How should researchers troubleshoot inconsistent NUP98 antibody staining patterns across different experimental conditions?

When troubleshooting inconsistent NUP98 antibody staining:

• Cell fixation and permeabilization variables:

  • Different fixation methods can affect epitope accessibility

  • For nuclear pore proteins, methanol fixation often preserves structure better than formaldehyde

  • Test multiple fixation protocols if inconsistent results are observed

• Cell cycle and physiological state considerations:

  • NUP98 distribution may vary across cell cycle stages due to nuclear envelope breakdown during mitosis

  • Synchronize cells or use cell cycle markers in co-staining to interpret heterogeneous patterns

• Nuclear extraction techniques:

  • For biochemical analyses, consider that different nuclear extraction protocols may selectively extract different NUP98 pools

  • Harsh extraction conditions may disrupt the nuclear pore complex and affect detection

• Antibody validation approaches:

  • Confirm specificity through siRNA knockdown controls

  • In double-immunofluorescence, verify expected co-localization with other known NPC components

  • For research involving specific domains, use truncation mutants to confirm epitope specificity

• Technical optimization strategies:

  • Titrate antibody concentration (1:50-1:500 for IF applications with 12329-1-AP)

  • Test multiple antigen retrieval methods for tissue sections (TE buffer pH 9.0 or citrate buffer pH 6.0)

  • Consider that the punctate nuclear rim pattern characteristic of NUP98 may require optimal microscopy settings for visualization

How can NUP98 antibodies be utilized in studying post-translational modifications and their impact on NUP98 function?

NUP98 antibodies can be instrumental in studying post-translational modifications (PTMs) through several approaches:

• Modification-specific detection:

  • Combine immunoprecipitation with NUP98 antibodies followed by western blotting with antibodies against specific modifications (phosphorylation, ubiquitination, SUMOylation)

  • Use phosphatase or deubiquitinase treatments as controls to confirm specificity of PTM detection

• Functional impact analysis:

  • Compare wild-type NUP98 and PTM-deficient mutants for:

    • RNA stabilization capacity using mRNA half-life assays

    • Protein-protein interactions through co-immunoprecipitation

    • Cellular localization via immunofluorescence microscopy

• Cell stress responses:

  • Investigate how DNA damage, oxidative stress, or other cellular stressors alter NUP98 modifications

  • This is particularly relevant given NUP98's roles in p53-mediated cellular responses, including offering partial protection from DNA damage-mediated apoptosis

• Methodological considerations:

  • Preserve modifications by including appropriate inhibitors in lysis buffers (phosphatase inhibitors, deubiquitinase inhibitors)

  • For low-abundance modifications, consider enrichment strategies before immunoblotting

What methodological approaches can be employed to study the structural dynamics of NUP98 within the nuclear pore complex?

To study NUP98 structural dynamics within the nuclear pore complex:

• Super-resolution microscopy techniques:

  • Use antibody-based STORM or PALM imaging to visualize NUP98 distribution at nanoscale resolution

  • Combine with other NPC component antibodies to map relative positioning

• Live-cell imaging approaches:

  • Engineer cell lines expressing fluorescent protein-tagged NUP98 validated by immunoblotting with NUP98 antibodies

  • Use FRAP (Fluorescence Recovery After Photobleaching) to assess mobility and exchange rates within the NPC

• Electron microscopy methods:

  • Immuno-gold electron microscopy with NUP98 antibodies reveals precise localization

  • Previous studies using double-immunoelectron microscopy have confirmed colocalization of TPR and Nup98 in the nuclear interior, with immunogold particles aligned in rows consistent with filamentous structures

• Interaction dynamics:

  • Study how NUP98 interactions with TPR and Nup96 contribute to nuclear architecture

  • Research has shown that Nup98 binds to TPR while the 6kDa peptide region mediates binding of Nup98 with Nup96

• Structural perturbation experiments:

  • Use domain-specific antibodies or competitive peptides to disrupt specific interactions

  • Previous work found that expression of myc-PK-6kDa fusion protein led to extensive staining of the TPR network, suggesting displacement of endogenous 6kDa peptide or Nup96

How can researchers integrate NUP98 antibody data with other omics approaches for comprehensive analyses?

Integrating NUP98 antibody data with other omics approaches:

• Multi-omics strategies:

  • Combine NUP98 ChIP-seq (for fusion proteins) with RNA-seq to correlate genomic binding with transcriptional outcomes

  • Integrate RNA immunoprecipitation data with proteomics to identify both RNA and protein partners

  • Use NUP98 antibodies for proximity labeling approaches (BioID, APEX) followed by mass spectrometry to map the NUP98 interaction network

• Systems biology frameworks:

  • Build interaction networks centered on NUP98 by combining antibody-based co-IP data with public interaction databases

  • Analyze how NUP98 network changes in disease contexts, particularly in hepatocellular carcinoma and leukemia where NUP98 functions have been implicated

• Technical challenges and solutions:

  • Address data normalization issues when comparing across different experimental platforms

  • Use appropriate statistical methods for integrating data from antibody-based experiments with high-throughput datasets

  • Validate key findings using orthogonal approaches to minimize technology-specific biases

• Translational research applications:

  • Correlate NUP98 expression or localization data with clinical outcomes

  • Explore potential of targeting NUP98 or its interaction partners as therapeutic strategies, particularly in contexts where NUP98 fusion proteins drive oncogenesis

What future developments can researchers anticipate in NUP98 antibody technologies and applications?

Anticipated future developments in NUP98 antibody technologies:

• Advanced antibody formats:

  • Domain-specific antibodies for distinguishing between different functional regions of NUP98

  • Recombinant nanobodies offering improved access to sterically hindered epitopes within the nuclear pore complex

  • Bifunctional antibodies for targeted degradation of pathogenic NUP98 fusion proteins

• Single-cell applications:

  • Adaptation of NUP98 antibodies for single-cell proteomics to capture cell-to-cell variation

  • Integration with spatial transcriptomics to correlate NUP98 localization with gene expression territories

• In vivo imaging:

  • Development of NUP98 antibody fragments suitable for intracellular delivery in live cells

  • PET imaging probes derived from NUP98 antibodies for non-invasive monitoring of NUP98 fusion proteins in leukemia models

• Therapeutic applications:

  • Antibody-drug conjugates targeting NUP98 fusion proteins in leukemia

  • Engineered antibody derivatives that can disrupt specific oncogenic interactions of NUP98 fusion proteins

• Technical innovations:

  • Multiplex imaging systems capable of simultaneously visualizing NUP98 alongside dozens of other nuclear factors

  • AI-assisted image analysis for quantifying subtle changes in NUP98 distribution patterns

  • Improved antibody validation methods ensuring reproducibility across experimental systems