NUP43 Antibody

What is NUP43 and why is it a significant research target?

NUP43 is a 43kDa nucleoporin protein that functions as a stable component of the Nup107-160 complex. It plays crucial roles in multiple biological processes, including positioning at the centromere during mitosis to regulate mitotic progression and chromosome segregation . NUP43 has gained significant research interest due to its involvement in the malignant transformation of cells and its overexpression in multiple cancers including gastric, breast, ovarian, and colorectal cancers . Recent studies have particularly highlighted its role in facilitating colorectal cancer development and liver metastasis, making it an important target for cancer research .

What are the primary applications of NUP43 antibody in research?

NUP43 antibodies are primarily utilized in three key research applications:

• Immunohistochemistry (IHC): For detecting and localizing NUP43 in tissue sections, commonly used at dilutions of 1:200-1:500 .

• Western Blotting (Immunoblotting): For protein expression analysis, typically used at concentrations of 0.04-0.4 μg/mL .

• Immunofluorescence (IF): For subcellular localization studies and co-localization experiments, particularly valuable for investigating nuclear translocation mechanisms .

These applications enable researchers to investigate NUP43 expression patterns, protein interactions, and functional roles in normal and pathological conditions.

How does NUP43 antibody specificity affect experimental outcomes?

Antibody specificity is crucial for accurate NUP43 research results. High-quality NUP43 antibodies, such as affinity-isolated polyclonal antibodies, provide superior specificity through thorough antigen region selection and stringent validation processes . The specificity can be verified through protein array testing against hundreds of human recombinant protein fragments, as implemented in validation protocols for Prestige Antibodies .

Poor antibody specificity can lead to false-positive signals, cross-reactivity with similar proteins, and misinterpretation of experimental data. Researchers should validate antibody specificity through appropriate controls including:

• Positive controls (tissues/cells known to express NUP43)

• Negative controls (tissues/cells with low/no NUP43 expression)

• Peptide competition assays to confirm binding specificity

• Correlation of results across multiple detection techniques (IHC, Western blot, IF)

How can NUP43 antibody be utilized to investigate protein-protein interactions in nuclear transport mechanisms?

NUP43 antibody can be employed in co-immunoprecipitation (Co-IP) assays to identify and characterize protein interaction networks involved in nuclear transport. This approach has revealed important insights into how NUP43 facilitates PD-L1 nuclear translocation through interaction with importin-5 (IPO5) .

Methodology for Co-IP with NUP43 antibody:

• Prepare protein lysates from cells of interest

• Incubate lysates with anti-NUP43, anti-PD-L1, anti-IPO5, anti-Flag, or anti-Myc antibodies at 4°C overnight with continuous rotation

• Add protein A/G beads or M2 anti-Flag resin and incubate at room temperature for 2-3 hours

• Wash beads three times with lysis buffer

• Elute bound proteins by boiling in SDS-PAGE sample buffer

• Analyze by western blot to detect interacting proteins

This technique has demonstrated that NUP43 enhances the nuclear translocation of PD-L1 by upregulating the PD-L1 binding protein IPO5, establishing a mechanistic link between NUP43 and nuclear PD-L1 accumulation in colorectal cancer .

What methodological approaches can be used to study NUP43's role in cancer progression using NUP43 antibody?

Investigating NUP43's role in cancer progression requires a multi-faceted approach:

• Expression Analysis in Clinical Samples:

  • Perform immunofluorescence double labeling with NUP43 and PD-L1 antibodies in cancerous and adjacent normal tissues

  • Compare expression levels and co-localization patterns between tumor and non-tumor tissues

  • Correlate expression with clinical outcomes using Kaplan-Meier survival analysis

• In Vitro Functional Studies:

  • Design specific shRNAs targeting NUP43 for gene silencing in cancer cell lines

  • Assess knockdown efficiency using qRT-PCR and western blotting

  • Conduct proliferation assays (CCK-8, EdU), invasion and migration assays (Transwell, scratch tests) to evaluate the impact of NUP43 modulation

• In Vivo Models:

  • Establish subcutaneous tumor-bearing mouse models using NUP43-knockdown and control cancer cells

  • Implement splenic injection models to assess liver metastasis

  • Analyze tumor size, weight, and metastatic burden

  • Perform immunohistochemical analysis for proliferation markers like Ki-67

These complementary approaches have demonstrated that NUP43 knockdown significantly inhibits tumor growth and colorectal cancer liver metastasis, suggesting its potential as a therapeutic target .

How can cellular fractionation be combined with NUP43 antibody analysis to investigate subcellular protein dynamics?

Cellular fractionation coupled with NUP43 antibody analysis provides valuable insights into the subcellular distribution and nuclear-cytoplasmic shuttling of proteins like PD-L1:

Methodology:

• Separate cellular fractions using a nuclear protein extraction kit

  • First lyse cells with cytoplasmic protein extraction buffer

  • Isolate the nuclear fraction using nuclear extraction buffer

• Confirm fraction purity using compartment-specific markers:

  • Tubulin (cytoplasmic marker)

  • Lamin B1 (nuclear marker)

• Perform western blot analysis on both fractions using:

  • Anti-NUP43 antibody

  • Anti-PD-L1 antibody

  • Additional antibodies for proteins of interest (e.g., IPO5, TM4SF1)

• Quantify protein levels in each fraction and calculate nuclear-to-cytoplasmic ratios

This approach has revealed that NUP43 overexpression increases nuclear PD-L1 (nPD-L1) levels, while NUP43 suppression significantly reduces nPD-L1 expression, demonstrating NUP43's role in facilitating PD-L1 nuclear translocation in colorectal cancer cells .

What are the optimal conditions for using NUP43 antibody in immunohistochemistry?

For optimal immunohistochemistry results with NUP43 antibody:

Sample Preparation:

• Fix tissues in 4% formaldehyde or 10% neutral buffered formalin

• Embed in paraffin and section at 4μm thickness

• Perform standard deparaffinization and rehydration

Antigen Retrieval:

• Heat-induced epitope retrieval in citrate buffer (pH 6.0) or EDTA buffer (pH 9.0)

• Microwave or pressure cooker treatment for 15-20 minutes

Immunostaining Protocol:

• Block endogenous peroxidase activity with 3% hydrogen peroxide

• Block non-specific binding with 2% bovine serum albumin (BSA) in PBS for 1 hour

• Incubate with primary NUP43 antibody at dilutions of 1:200-1:500 overnight at 4°C

• Apply appropriate HRP-conjugated secondary antibody for 1 hour at room temperature

• Develop with 3,3'-diaminobenzidine (DAB)

• Counterstain with hematoxylin

• Mount and analyze under a light microscope

Controls:

• Include positive controls (tissues known to express NUP43)

• Include negative controls (primary antibody omitted)

• Consider double-staining with other markers of interest (e.g., PD-L1)

How should researchers design quantitative experiments using NUP43 antibody for western blotting?

Designing quantitative western blot experiments with NUP43 antibody requires careful planning:

Sample Preparation:

• Extract proteins using appropriate lysis buffers containing protease inhibitors

• Determine protein concentration using Bradford or BCA assay

• Load equal amounts of protein (typically 20-40μg) per lane

Electrophoresis and Transfer:

• Separate proteins on 10-12% SDS-PAGE gels

• Transfer to PVDF or nitrocellulose membranes

Immunoblotting Protocol:

• Block membranes with 5% non-fat milk or BSA in TBST

• Incubate with primary NUP43 antibody at 0.04-0.4 μg/mL overnight at 4°C

• Wash thoroughly with TBST (3-5 times, 5-10 minutes each)

• Incubate with appropriate HRP-conjugated secondary antibody

• Develop using enhanced chemiluminescence (ECL) detection

Quantification and Normalization:

• Use housekeeping proteins (GAPDH, Tubulin) as loading controls

• For nuclear fraction analysis, use Lamin B1 as a nuclear loading control

• Capture images within the linear range of detection

• Quantify band intensities using ImageJ or similar software

• Calculate relative expression as the ratio of NUP43 to loading control

Experimental Design Considerations:

• Include technical replicates (2-3 per sample)

• Perform biological replicates (minimum 3 independent experiments)

• Include appropriate positive and negative controls

• Consider time-course or dose-response studies when relevant

What is the recommended protocol for dual immunofluorescence staining with NUP43 and other protein markers?

For dual immunofluorescence staining with NUP43 antibody and other markers (e.g., PD-L1):

Sample Preparation:

• For tissue sections: Cut paraffin-embedded tissues into 4μm sections, deparaffinize, and rehydrate

• For cells: Culture on coverslips, fix with 4% formaldehyde for 20 minutes at room temperature

Permeabilization and Blocking:

• Permeabilize with 0.05% Triton X-100 in PBS for 5 minutes

• Block with 2% BSA in PBS for 1 hour at room temperature

Primary Antibody Incubation:

• Prepare a mixture of anti-NUP43 antibody (rabbit) and second marker antibody (different host species)

• Incubate samples with the antibody mixture overnight at 4°C

• Wash three times with PBS (5 minutes each)

Secondary Antibody Incubation:

• Use species-specific Alexa Fluor-conjugated secondary antibodies with distinct fluorophores

• Incubate for 1 hour at room temperature in the dark

• Wash three times with PBS (5 minutes each)

Nuclear Counterstaining and Mounting:

• Counterstain nuclei with DAPI

• Mount slides with anti-fade mounting medium

• Seal with nail polish

Imaging and Analysis:

• Capture images using confocal microscopy

• Analyze co-localization using appropriate software (ImageJ with co-localization plugins)

• Quantify signal intensity and calculate Pearson's correlation coefficient for co-localization analysis

This protocol has successfully demonstrated co-localization of NUP43 with PD-L1 in colorectal cancer tissues and cell lines .

How can researchers address non-specific binding when using NUP43 antibody?

Non-specific binding is a common challenge when working with antibodies. For NUP43 antibody, consider these troubleshooting strategies:

Causes and Solutions for Non-specific Binding:

Validation Approaches:

• Perform protein array testing against multiple human recombinant protein fragments

• Include appropriate positive and negative controls

• Compare results with alternative detection methods

• Consider using knockout/knockdown samples as negative controls

How should researchers interpret conflicting results between different detection methods using NUP43 antibody?

When facing discrepancies between different detection methods:

• Consider Method-Specific Limitations:

  • Western blot detects denatured proteins and may miss conformational epitopes

  • IHC preserves tissue context but may have lower sensitivity

  • IF provides subcellular localization but may be affected by fixation artifacts

• Systematic Validation Approach:

  • Verify antibody specificity in each method independently

  • Test multiple antibody concentrations and incubation conditions

  • Use knockout/knockdown controls in each method

  • Consider method-specific sample preparation impacts on epitope accessibility

• Reconciliation Strategies:

  • Cross-validate results with alternative antibody clones or epitopes

  • Use complementary techniques like mass spectrometry

  • Correlate findings with functional data (e.g., gene expression, phenotypic assays)

  • Consider subcellular fractionation to verify localization findings

• Data Integration Framework:

  • Prioritize consistency across biological replicates over technical variations

  • Consider the biological context and existing literature

  • Weight evidence based on technical robustness of each method

  • Report all findings transparently, acknowledging limitations

Research has shown that NUP43 facilitates PD-L1 nuclear translocation, which was confirmed through multiple complementary techniques including immunofluorescence, western blotting of nuclear/cytoplasmic fractions, and co-immunoprecipitation, demonstrating the value of method triangulation .

What strategies can researchers employ to quantitatively analyze nuclear versus cytoplasmic localization of proteins using NUP43 antibody?

Quantitative analysis of nuclear versus cytoplasmic protein localization is crucial for understanding NUP43's role in nuclear transport:

Cellular Fractionation and Western Blot Approach:

• Perform cellular fractionation as described previously

• Normalize nuclear protein levels to Lamin B1 and cytoplasmic levels to Tubulin

• Calculate nuclear-to-cytoplasmic ratio for proteins of interest

• Compare ratios across experimental conditions (e.g., NUP43 overexpression vs. control)

Image Analysis of Immunofluorescence Data:

• Capture high-resolution confocal microscopy images

• Define nuclear regions using DAPI staining as a mask

• Define cytoplasmic regions by subtracting nuclear mask from whole-cell outline

• Measure mean fluorescence intensity in each compartment

• Calculate nuclear-to-cytoplasmic intensity ratios

• Analyze multiple cells (n≥50) per condition for statistical reliability

• Apply appropriate statistical tests (t-test, ANOVA) to compare conditions

Software Tools:

• ImageJ with Nuclear-Cytoplasmic Ratio plugin

• CellProfiler with compartmental analysis modules

• MATLAB with custom image analysis scripts

This approach has successfully demonstrated that NUP43 overexpression significantly increases the nuclear localization of PD-L1 in colorectal cancer cells, providing quantitative evidence for NUP43's role in nuclear transport mechanisms .

How does NUP43's role in the PD-L1/nPD-L1/PD-L1 feedback loop impact cancer immunotherapy research?

Recent research has uncovered a critical role for NUP43 in modulating the PD-L1/nPD-L1/PD-L1 feedback loop, with significant implications for cancer immunotherapy:

Mechanism of Action:

• NUP43 facilitates the nuclear translocation of PD-L1 (creating nuclear PD-L1 or nPD-L1) by upregulating the PD-L1 binding protein IPO5

• nPD-L1 participates in a feedback loop that further enhances PD-L1 expression

• Elevated membrane PD-L1 contributes to immune evasion in colorectal cancer

Immunotherapy Implications:

• Traditional anti-PD-L1/PD-1 therapies target membrane-bound PD-L1 but may not address the nPD-L1-mediated feedback mechanism

• Targeting NUP43 could potentially disrupt this feedback loop, enhancing the efficacy of immunotherapy

• Combined approaches targeting both membrane PD-L1 and the NUP43-mediated nuclear transport pathway may provide more comprehensive immune checkpoint blockade

Research Directions:

• Develop specific inhibitors of NUP43-IPO5 interaction

• Investigate the correlation between NUP43 expression and response to PD-1/PD-L1 inhibitors in clinical samples

• Explore combination therapies targeting both NUP43 and PD-1/PD-L1 pathways

• Study the broader impact of nuclear PD-L1 on the tumor microenvironment

This understanding of the PD-L1-nPD-L1-PD-L1 feedback loop provides a novel therapeutic strategy for colorectal cancer patients, potentially extending to other cancer types where NUP43 is overexpressed .

What experimental approaches can researchers use to investigate the relationship between NUP43 and cancer metastasis?

Investigating NUP43's role in cancer metastasis requires sophisticated experimental approaches:

In Vitro Models:

• Invasion and Migration Assays:

  • Transwell assays with or without Matrigel coating

  • Wound healing/scratch assays

  • 3D organoid invasion models

  • Compare NUP43 knockdown/overexpression cells with controls

• Epithelial-Mesenchymal Transition (EMT) Analysis:

  • Monitor EMT markers (E-cadherin, N-cadherin, Vimentin) in NUP43-modulated cells

  • Assess morphological changes using phase-contrast microscopy

  • Analyze cytoskeletal reorganization through F-actin staining

In Vivo Metastasis Models:

• Splenic Injection Model:

  • Inject NUP43-knockdown or control cancer cells into mouse spleens

  • Monitor liver metastasis development

  • Quantify metastatic burden through imaging and histological analysis

  • Perform immunohistochemistry for proliferation markers like Ki-67

• Tail Vein Injection Model:

  • Assess lung metastatic potential

  • Compare metastatic colonization efficiency between NUP43-modulated and control cells

• Orthotopic Implantation:

  • Implant cells directly into the organ of origin (e.g., colon)

  • Monitor natural metastatic spread

  • Correlate with NUP43 expression levels

Clinical Correlation Studies:

• Analyze NUP43 expression in primary tumors versus matched metastatic lesions

• Correlate NUP43 levels with metastasis-free survival

• Investigate associations between NUP43 expression and established metastasis biomarkers

These approaches have demonstrated that NUP43 knockdown significantly inhibits colorectal cancer liver metastasis in mouse models, suggesting its potential as a therapeutic target for metastatic disease .

What are the current technical limitations of NUP43 antibody-based research and emerging solutions?

Current technical limitations and emerging solutions in NUP43 antibody-based research include:

Current Limitations:

• Antibody Specificity Challenges:

  • Cross-reactivity with related nucleoporins

  • Potential recognition of non-specific epitopes

  • Variability between antibody lots

• Detection Sensitivity Issues:

  • Limited sensitivity for detecting low abundance proteins

  • Difficulties in quantifying small changes in expression or localization

  • Background signal in certain tissue types

• Dynamic Process Visualization:

  • Static nature of traditional antibody-based methods

  • Inability to track real-time protein movements

  • Limited information about protein-protein interaction dynamics

Emerging Solutions:

• Advanced Antibody Technologies:

  • Development of super-resolution microscopy-compatible antibodies

  • Nanobodies and single-domain antibodies for improved tissue penetration

  • Recombinant antibody fragments with enhanced specificity

• Complementary Methodologies:

  • CRISPR-based tagging of endogenous NUP43

  • Proximity ligation assays for improved detection of protein interactions

  • Mass spectrometry validation of antibody-based findings

• Live-Cell Imaging Approaches:

  • Fluorescent protein fusions to monitor NUP43 dynamics

  • FRAP (Fluorescence Recovery After Photobleaching) for nuclear transport kinetics

  • Single-molecule tracking methods

• Computational Methods:

  • Machine learning algorithms for improved image analysis

  • Integrative multi-omics approaches combining antibody-based data with genomics and proteomics

  • Systems biology modeling of nuclear transport processes

These advances will enhance our ability to investigate NUP43's complex roles in nuclear transport and cancer progression, potentially leading to novel therapeutic strategies targeting the NUP43-mediated processes involved in disease .