NUP93A Antibody

What is NUP93 and why is it significant in cellular biology?

NUP93 is a critical component of the nuclear pore complex (NPC) that plays essential roles in nuclear pore assembly and maintenance . Beyond its structural function, NUP93 regulates podocyte migration and proliferation through SMAD4 signaling during renal development . Recent research has revealed its involvement in cancer progression, particularly in aggressive breast cancer subtypes, where it facilitates nucleocytoplasmic transport of transcription factors . NUP93's functional versatility makes it an important target for both basic cell biology and disease-focused research.

Which applications are NUP93 antibodies validated for?

Commercial NUP93 antibodies have been validated for multiple applications, with validation specifications varying by manufacturer:

When selecting an antibody, consider both the validated applications and the specific epitope recognized, as this affects detection of different protein domains or isoforms.

How should I determine the appropriate dilution for NUP93 antibodies?

Optimal antibody dilution requires systematic titration to achieve maximum signal-to-noise ratio. Start with the manufacturer's recommended dilution ranges (e.g., 1:500-1:2,000 for Western blot ), then perform a dilution series in your specific experimental system. For Western blotting, use a dilution series (e.g., 1:500, 1:1000, 1:2000) on lysates known to express NUP93. For immunohistochemistry, consider testing a broader range (e.g., 1:100-1:400) . Always include appropriate controls and evaluate both signal intensity and background levels. Document your optimization process for reproducibility and consider that antibody performance may vary between batches.

How can I optimize NUP93 detection in triple-negative breast cancer samples?

Triple-negative breast cancer (TNBC) samples require specialized optimization for NUP93 detection due to the critical role of NUP93 in this aggressive cancer subtype . Research has shown that NUP93 overexpression is independently associated with shorter disease-free survival in TNBC . For optimal detection:

• Antigen retrieval: Use high-pressure citrate buffer (pH 6.0) pretreatment for formalin-fixed paraffin-embedded samples

• Blocking: Block with 10% normal goat serum for 30 minutes at room temperature before primary antibody incubation

• Antibody incubation: Dilute primary antibody in 1% BSA and incubate at 4°C overnight

• Detection system: Use a biotinylated secondary antibody with an HRP-conjugated system for visualization

• Controls: Include normal breast tissue samples and known NUP93-positive TNBC samples as controls

Additionally, considering that NUP93 expression correlates with the basal subtype of breast cancer, use MDA-MB-231 cells as positive controls for antibody validation .

What are the best fixation methods for preserving NUP93 epitopes?

Preserving NUP93 epitopes requires careful consideration of fixation methods, as nuclear pore complex proteins can be sensitive to fixation artifacts:

• Paraformaldehyde fixation: 4% paraformaldehyde for 15-20 minutes at room temperature preserves most NUP93 epitopes while maintaining cellular architecture

• Methanol fixation: Ice-cold methanol (-20°C) for 10 minutes is suitable for revealing certain nuclear epitopes but may disrupt membrane structures

• Combination fixation: For some applications, a brief paraformaldehyde fixation (10 minutes) followed by methanol permeabilization may provide optimal epitope accessibility

When using commercially available NUP93 antibodies for immunohistochemistry, high-pressure antigen retrieval in citrate buffer (pH 6.0) has been demonstrated to effectively retrieve epitopes in formalin-fixed paraffin-embedded tissue samples . Always validate your fixation protocol with appropriate controls, including known NUP93-expressing cell lines like MDA-MB-231 .

How do I resolve non-specific binding issues with NUP93 antibodies?

Non-specific binding can compromise NUP93 antibody specificity. Address this common challenge through:

• Fc receptor blocking: Pre-incubate samples with species-appropriate serum or commercial Fc blocking reagents

• Titration optimization: Systematically test dilution series to find the concentration that maximizes signal-to-noise ratio

• Alternative blocking agents: Test different blocking solutions (5% BSA, 5% milk, commercial blockers) to determine which minimizes background

• Secondary antibody optimization: Ensure secondary antibodies are highly cross-adsorbed against other species

• Antibody validation: Confirm specificity using knockdown/knockout controls. Research has confirmed specificity using RNAi-resistant NUP93 expression as a control

For Western blotting applications, the predicted molecular weight of NUP93 is 93 kDa, but some antibodies detect bands around 72 kDa . These differences may represent post-translational modifications or isoforms. Always include positive and negative controls to confirm specificity.

What controls should I include when studying NUP93 in experimental systems?

Rigorous controls are essential for interpreting NUP93 studies. Include:

• Positive controls: Cell lines with verified NUP93 expression (e.g., MDA-MB-231 for basal breast cancer studies)

• Negative controls: Samples with NUP93 knockdown using validated siRNAs or shRNAs

• Expression rescue controls: RNAi-resistant NUP93 expression can rescue phenotypes in knockdown experiments, confirming specificity

• Isotype controls: For immunostaining, include appropriate isotype-matched control antibodies

• Secondary-only controls: Samples incubated with secondary antibody alone to assess non-specific binding

For flow cytometry applications, fluorescence minus one (FMO) controls are recommended to establish proper gating strategies . When analyzing NUP93 expression in tissue samples, include both normal adjacent tissue and known positive samples for comparison.

How can I design experiments to investigate NUP93's role in cancer progression?

Based on recent research findings, consider these experimental approaches:

• Cell proliferation assays: Measure DNA synthesis using radioactive nucleoside incorporation in cells with modulated NUP93 expression

• Cell viability assessment: Compare viability in cells with inducible NUP93 overexpression or knockdown

• Migration and invasion assays: Evaluate whether NUP93 affects cell motility through actin cytoskeleton remodeling

• In vivo tumor models: Implant cells with tetracycline-inducible NUP93 expression in mouse mammary fat pads to measure tumor growth and metastasis

• Lung colonization assays: Inject cells with modulated NUP93 expression into tail veins to assess metastatic potential

Research has shown that NUP93 overexpression enhances tumor growth and metastasis in mouse models, while NUP93 depletion reduces these effects . These phenotypes appear to be specific to NUP93, as siRNAs targeting other nuclear pore components (NUP62, NUP98, NUP107) did not produce the same effects .

How is NUP93 expression correlated with breast cancer outcomes?

NUP93 expression is significantly associated with breast cancer prognosis, particularly in aggressive subtypes:

• Triple-negative and HER2-positive tumors: High NUP93 expression correlates with shorter disease-free survival in these subtypes

• Estrogen receptor status: ER-negative tumors, which are generally more aggressive, display relatively high NUP93 expression

• Integrative clusters: Among the ten integrative clusters of breast cancer, IC10 (which includes mostly triple-negative tumors) shows the highest NUP93 expression

These findings suggest that NUP93 could serve as a prognostic marker and potential therapeutic target in aggressive breast cancer subtypes. The correlation between NUP93 expression and poor prognosis appears to be independent of other factors, suggesting a mechanistic role in disease progression rather than merely an association .

What mechanisms connect NUP93 to cell migration and metastasis?

Recent research has uncovered a novel connection between NUP93 and the actin cytoskeleton:

• Actin cytoskeleton remodeling: NUP93 modulates cell migration through actin cytoskeleton (AC) remodeling

• Stress fiber regulation: Depletion of NUP93 causes a dramatic decrease in actin stress fibers, as demonstrated by reduced staining of the stress fiber-associated protein LIMCH1

• Nuclear-cytoskeletal coupling: NUP93 may facilitate interactions between the nuclear lamina and the actin cytoskeleton through adaptor proteins

• Specificity: This function appears to be specific to NUP93, as silencing other NPC components does not produce the same phenotype

The connection between NUP93 and actin cytoskeleton dynamics represents an important mechanistic link between nuclear pore complex function and cancer cell migration/invasion. Targeting this interaction could represent a novel therapeutic approach for metastatic cancer .

How do experimental approaches for studying NUP93 differ from other nuclear pore proteins?

Studying NUP93 requires specific experimental approaches due to its unique functions:

• Inducible expression systems: Tetracycline/doxycycline-inducible systems for NUP93 overexpression or knockdown allow controlled modulation of protein levels

• RNAi-resistant constructs: Expression of RNAi-resistant NUP93 variants provides crucial controls for knockdown specificity

• Cytoskeletal analysis: Unlike other nuclear pore proteins, NUP93 studies should include assessment of actin cytoskeleton organization

• Metastasis assays: In vivo models specifically examining metastatic potential are particularly relevant for NUP93 research

• Nuclear pore density analysis: Electron microscopy and immunofluorescence analysis of nuclear pore density provides important insights into NUP93 function

When designing experiments to study NUP93, researchers should consider both its canonical roles in nuclear pore complex assembly and its non-canonical functions in signaling and cytoskeletal regulation. This dual functionality makes NUP93 a particularly interesting target for cancer research.

What are the differences between polyclonal and monoclonal NUP93 antibodies?

When selecting between polyclonal and monoclonal NUP93 antibodies, consider these comparative factors:

Polyclonal antibodies may offer advantages for detection of low-abundance targets or when protein conformation might mask certain epitopes. Monoclonal antibodies provide higher specificity and reproducibility. Recombinant antibodies combine specificity with consistent production without animal use .

How should I validate NUP93 antibody specificity for my experimental system?

Thorough validation is critical before using NUP93 antibodies in research:

• Knockdown/knockout controls: Use siRNA, shRNA, or CRISPR to deplete NUP93 and confirm signal reduction with the antibody

• Overexpression validation: Express tagged NUP93 and confirm co-localization with antibody signal

• Cross-reactivity assessment: Test antibody on samples from multiple species if cross-reactivity is claimed

• Alternative antibodies: Compare results using antibodies targeting different epitopes of NUP93

• Published validation: Check literature for previous validation in similar experimental systems

For Western blotting, confirm that the detected band matches the predicted molecular weight (93 kDa) . Observed molecular weights may vary (some detect at 72 kDa) due to post-translational modifications or splice variants . For immunostaining, include subcellular localization controls, as NUP93 should primarily localize to the nuclear envelope in a punctate pattern characteristic of nuclear pore complexes.