NUP93 Antibody, FITC conjugated

What is NUP93 and why is it important in cell biology?

NUP93 is a key structural component of the nuclear pore complex (NPC), primarily localized to the nuclear side, particularly within the basket terminal ring area. It forms critical complexes with other nucleoporins including Nup188, Nup53, and Nup205, which are essential for NPC stability and assembly . NUP93 constitutes over 10% of the total NPC mass, making it the most abundant nucleoporin . Structurally, NUP93 contains a coiled-coil domain at its N-terminus and a C-terminal helical domain, both crucial for its functional integrity . The protein plays a vital role in mediating the transport of macromolecules between the nucleus and cytoplasm, which is essential for maintaining cellular function and homeostasis . Disruptions in NUP93 activity can lead to impaired nucleocytoplasmic transport and subsequent cellular dysfunction, highlighting its biological significance .

What species reactivity does NUP93 Antibody, FITC conjugated support?

NUP93 Antibody (F-2), available in FITC-conjugated form, has been validated to detect NUP93 in mouse, rat, and human samples . This cross-species reactivity makes it valuable for comparative studies across different model organisms. The antibody is a mouse monoclonal IgG2b kappa light chain antibody that recognizes specific epitopes of NUP93 across these species . Similarly, the E-8 variant (also available with various conjugates) detects NUP93 protein of mouse, rat, and human origin, providing researchers with options depending on their experimental needs .

What cell types and tissues express NUP93?

NUP93 expression has been documented in multiple cell types and tissues. Immunofluorescence analysis has detected NUP93 expression in nuclei of all glomerular and tubulointerstitial cells in human kidneys . In kidney samples, NUP93 signals are detected in cells both inside and outside of glomeruli, confirming its widespread expression . Additionally, NUP93 has been studied in endothelial cells (ECs), where its expression levels affect cellular senescence and reprogramming toward healthy phenotypes . NUP93 is also expressed in keratinocytes, where its downregulation modulates differentiation processes . The universal expression of NUP93 across diverse cell types reflects its fundamental role in nuclear transport and cellular function.

What are the optimal applications for NUP93 Antibody, FITC conjugated?

NUP93 Antibody, FITC conjugated is suitable for multiple experimental applications. The F-2 antibody has been validated for western blotting (WB), immunoprecipitation (IP), immunofluorescence (IF), and enzyme-linked immunosorbent assay (ELISA) . The FITC conjugation particularly enhances its utility for immunofluorescence applications, eliminating the need for secondary antibody incubation. For immunofluorescence studies, researchers have successfully used anti-NUP93 antibodies at dilutions of 1:500 for tissue sections . For Western blot applications, anti-NUP93 antibodies have been effectively used at 1:1000 dilution . When designing experiments with this antibody, researchers should consider that NUP93 primarily localizes to the nuclear envelope in a punctate pattern characteristic of nuclear pore complexes, which should inform expected staining patterns.

How can NUP93 Antibody, FITC conjugated be used in tissue immunofluorescence studies?

For tissue immunofluorescence studies, protocols have been established for effective NUP93 detection. Tissues should first be fixed with 4% PFA/PBS solution (4°C, overnight) followed by either paraffin embedding or dehydration in 30% sucrose/PBS solution for OCT embedding . After sectioning (typically 5 μm), samples should be permeabilized with 0.2% Triton-X 100/PBS for 10 minutes at room temperature and blocked with 5% BSA/PBS for 1 hour . The NUP93 Antibody, FITC conjugated can then be applied at appropriate dilutions (e.g., 1:500) and incubated overnight at 4°C . For dual immunofluorescence assays, NUP93 antibodies have been successfully paired with markers such as CD2AP and nephrin to study glomerular expression patterns . When optimizing staining protocols, researchers should consider including preabsorption controls to confirm staining specificity, as demonstrated in previous studies where signals were significantly decreased when antibody was preabsorbed with immunoprecipitates from cells expressing FLAG-tagged NUP93 .

What controls should be used when working with NUP93 Antibody, FITC conjugated?

Appropriate controls are essential for validating results with NUP93 Antibody, FITC conjugated. Negative controls should include samples incubated with isotype-matched control antibodies to assess non-specific binding. For preabsorption controls, the antibody should be preincubated with immunoprecipitates from cells expressing tagged NUP93 before application to samples . This approach has been shown to significantly decrease staining intensity, confirming antibody specificity . Positive controls should include tissues or cells known to express NUP93, such as kidney sections or HEK293 cells expressing NUP93 . For knockdown validation studies, comparing staining intensity between control samples and those with NUP93 knockdown can demonstrate specificity, as NUP93 knockdown results in decreased nuclear staining intensity . Additionally, Western blot analysis can be performed in parallel to confirm antibody specificity before immunofluorescence studies.

How can NUP93 Antibody, FITC conjugated be used to study NPC assembly mechanisms?

NUP93 Antibody, FITC conjugated provides a valuable tool for investigating nuclear pore complex (NPC) assembly mechanisms. Research has established that the C-terminal domain of NUP93 (amino acids 608-820) is necessary and sufficient for forming a minimal structural NPC backbone . Using this antibody in conjunction with reconstitution experiments, researchers can examine how different domains of NUP93 contribute to NPC assembly. For instance, studies have shown that while the C-terminal fragment allows for formation of a closed nuclear envelope, the resulting NPCs show reduced levels of FG-containing nucleoporins . Time-course immunofluorescence studies during nuclear assembly can reveal the sequential recruitment of NUP93 and its interaction partners. By combining NUP93 antibody staining with antibodies against other nucleoporins like the Nup107-160 complex or Nup98, researchers can map the hierarchical assembly process of NPCs . Such studies are critical for understanding the fundamental mechanisms of nuclear envelope formation and NPC biogenesis.

How can NUP93 Antibody be utilized in studies of kidney diseases related to NUP93 mutations?

NUP93 mutations have been reported to cause steroid-resistant nephrotic syndrome (SRNS) and focal segmental glomerulosclerosis (FSGS), often progressing to end-stage renal disease in childhood . The NUP93 Antibody can be instrumental in studying these pathological conditions. Immunofluorescence analysis using this antibody has revealed that in patients with compound heterozygous NUP93 mutations (e.g., p.Arg525Trp and p.Tyr629Cys), the intensity of NUP93 immunofluorescence is significantly decreased in nuclei of both glomerular and extraglomerular cells . By comparing the expression patterns of NUP93 in normal versus disease samples, researchers can gain insights into the molecular mechanisms of these kidney disorders. Dual immunofluorescence studies combining NUP93 with podocyte markers like CD2AP and nephrin have shown that while NUP93 expression is decreased in patient samples, glomerular expression of CD2AP remains relatively intact and nephrin is only partially decreased in sclerotic lesions . These findings suggest that NUP93 mutations may affect kidney function through mechanisms beyond disruption of podocyte proteins, opening new avenues for research into SRNS and FSGS pathogenesis.

What approaches can be used to study NUP93's role in cellular differentiation and aging?

NUP93 has emerging roles in cellular differentiation and aging that can be investigated using the FITC-conjugated antibody. In keratinocytes, NUP93 knockdown has been shown to induce differentiation genes related to both mechanical and immune barrier functions, including activation of NF-κB target genes . Researchers can use NUP93 Antibody, FITC conjugated to track changes in NUP93 expression and localization during differentiation processes. For aging studies, experiments have revealed that restoring NUP93 expression in senescent endothelial cells (ECs) reverses aging and promotes endothelial reprogramming toward a healthy phenotype . Combining immunofluorescence for NUP93 with senescence markers (e.g., γH2AX) can provide insights into the relationship between nuclear pore complex composition and cellular aging. Functional assays can complement these studies, as NUP93 knockdown has been shown to diminish keratinocytes' clonogenicity and epidermal regenerative capacity . For quantitative assessment, keratinocyte competition assays where GFP-labeled control cells compete with DsRed-labeled NUP93 knockdown cells can evaluate the impact on tissue regeneration .

What might cause weak or absent staining when using NUP93 Antibody, FITC conjugated?

Several factors can contribute to weak or absent staining when using NUP93 Antibody, FITC conjugated. Insufficient permeabilization is a common issue, as NUP93 is located within the nuclear pore complex structure, which may require optimal permeabilization conditions (typically 0.2% Triton-X 100/PBS for 10 minutes) . Inadequate fixation can also impact antibody accessibility to the antigen; tissues should be properly fixed with 4% PFA/PBS solution overnight at 4°C . The storage conditions of the antibody itself are critical; FITC conjugates are particularly sensitive to light exposure and may lose fluorescence intensity if improperly stored. If working with paraffin-embedded tissues, incomplete antigen retrieval can hinder antibody binding. For biological reasons, certain disease states or experimental conditions may genuinely result in reduced NUP93 expression, as observed in patients with NUP93 mutations where the intensity of NUP93 immunofluorescence is significantly decreased . To troubleshoot, researchers should include positive control samples with known NUP93 expression, optimize permeabilization and fixation protocols, and ensure proper antibody handling and storage.

How should contradictory results between NUP93 immunofluorescence and other detection methods be reconciled?

When faced with contradictory results between NUP93 immunofluorescence and other detection methods, researchers should consider several factors. First, antibody epitope accessibility may differ between applications; the F-2 antibody detects specific epitopes that might be masked in certain experimental conditions or preparations . In one study, although NUP93 immunofluorescence was significantly decreased in a patient's kidney tissue, the amount of NUP93 protein was not significantly altered in the patient's peripheral blood mononuclear cells as detected by Western blot . This discrepancy highlights tissue-specific effects or differences in detection sensitivity between methods. Different antibody clones (such as F-2 vs. E-8) may recognize different epitopes with varying accessibility across techniques . To reconcile such contradictions, researchers should: 1) Verify antibody specificity using knockdown/knockout controls in the specific application; 2) Compare results using alternative antibody clones targeting different epitopes; 3) Consider that post-translational modifications might affect epitope recognition in different assays; 4) Assess potential differences in protein localization versus total expression levels that might explain discrepancies between immunofluorescence (location-specific) and Western blot (total protein) results.

How might NUP93 Antibody be used to investigate the role of nuclear pore complexes in gene regulation?

NUP93 Antibody, FITC conjugated offers promising applications for studying the emerging role of nuclear pore complexes in gene regulation. Recent research has linked NUP93 knockdown to the induction of differentiation genes and increased nuclear localization of NF-κB p65/p50 transcription factors in keratinocytes . To investigate these regulatory mechanisms, researchers can combine NUP93 immunofluorescence with transcription factor localization studies. Chromatin immunoprecipitation (ChIP) experiments following NUP93 knockdown or overexpression can identify genomic regions affected by changes in NUP93 levels. Dual immunofluorescence for NUP93 and chromatin markers or transcription factors can reveal potential associations between NPC components and chromatin organization. Additionally, reporter assays measuring transcriptional activity (such as NF-κB reporter assays) in conjunction with NUP93 manipulation can provide functional evidence for its role in gene regulation . These approaches can help elucidate how structural components of the nuclear pore complex contribute to gene expression control, potentially revealing novel regulatory mechanisms that extend beyond the traditional transport functions of nucleoporins.

What is the potential for using NUP93 as a marker in studies of cellular stress responses?

NUP93 has emerging potential as a marker in cellular stress response studies. Research has demonstrated that NUP93 expression levels can change in response to cellular stressors, and these changes may have functional consequences. For instance, in endothelial cells, the restoration of NUP93 expression in senescent cells can reverse aging phenotypes, suggesting a role in stress-induced senescence pathways . Using the FITC-conjugated NUP93 antibody, researchers can track changes in NUP93 localization or expression levels following exposure to various stressors such as oxidative stress, DNA damage, or inflammatory stimuli. Co-staining for stress markers like γH2AX (a marker for DNA damage) alongside NUP93 can reveal correlations between nuclear pore complex alterations and cellular stress responses . This approach may be particularly valuable in age-related disease models, where nuclear pore dysfunction has been implicated in pathogenesis. Furthermore, time-course studies examining NUP93 dynamics during stress recovery could provide insights into the role of nuclear transport regulation in cellular resilience and adaptation.