NUP93B Antibody

What is NUP93 and why is it an important research target?

NUP93 is a 93 kDa nucleoporin that forms part of the nuclear pore complex (NPC), playing a crucial role in nuclear envelope architecture and nucleocytoplasmic transport. It's significant in research for several reasons:

• It functions as a structural component of the NPC core and contributes to NPC assembly and maintenance

• It forms complexes with other nucleoporins including NUP53, NUP155, NUP205, and lamin B

• NUP93 expression inversely correlates with survival in triple-negative breast cancer patients

• Mutations in NUP93 have been linked to steroid-resistant nephrotic syndrome (SRNS) and focal segmental glomerulosclerosis (FSGS)

When designing experiments targeting NUP93, researchers should consider its subcellular localization (predominantly in the nuclear membrane) and its interactions within larger protein complexes.

What applications are NUP93 antibodies validated for?

NUP93 antibodies have been validated for multiple applications with specific working dilutions:

For IHC-Paraffin applications, heat-induced epitope retrieval (HIER) at pH 6 is recommended for optimal results . The choice of application should be guided by your specific research question and experimental design.

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

Validating antibody specificity is crucial for generating reliable data. For NUP93 antibodies, consider these approaches:

• Genetic validation: Use cells with NUP93 knockdown/knockout as negative controls. Research has demonstrated specificity through reduced signal in NUP93-depleted samples .

• Antibody absorption test: As demonstrated in kidney tissue studies, pre-absorb the antibody with immunoprecipitates from cells expressing FLAG-tagged NUP93, which should significantly decrease signal intensity if the antibody is specific .

• Molecular weight verification: Confirm detection of a band at the expected 93 kDa size in Western blot .

• Cross-validation: Compare results using multiple NUP93 antibodies targeting different epitopes. Available antibodies target various regions including the N-terminal 300 amino acids , amino acids 221-270 , and regions between amino acids 575-625 .

• RNAi-resistant rescue: Express an RNAi-resistant version of NUP93 in depleted cells to demonstrate signal recovery, as shown in MDA-MB-231 cells .

What considerations are important when studying NUP93 in kidney tissue samples?

When investigating NUP93 in kidney tissues, especially in the context of nephrotic syndrome research:

• Cell type specificity: NUP93 is expressed in nuclei of all glomerular and tubulointerstitial cells in human kidneys. Use double-immunofluorescence with cell-specific markers for accurate interpretation:

  • CD2AP for podocytes

  • CD31 for endothelial cells

  • Amnionless for proximal tubules

• Signal pattern: Normal NUP93 expression appears as dots in nuclei of kidney cells, with signals detected in both CD2AP-positive (podocytes) and CD2AP-negative cells within glomeruli .

• Mutation effects: In patients with NUP93 mutations, observe for diffuse decrease in NUP93 staining intensity across both glomerular and extraglomerular cells, while monitoring other markers like CD2AP and nephrin for comparison .

• Control selection: Use donor kidneys as positive controls to establish baseline expression patterns .

This methodological approach enables differentiation between normal and pathological NUP93 expression patterns in kidney tissues.

How can NUP93 antibodies be utilized to study its role in cancer progression?

NUP93 has significant implications in cancer research, particularly in breast cancer. When designing experiments:

• Invasion and migration assays: NUP93 depletion decreases breast cancer cell invasion through 3D matrices by approximately 33% (66.9% ± 6.0% versus 100.0% ± 7.8% in controls) . Use NUP93 antibodies to confirm knockdown efficiency before functional assays.

• Cytoskeletal studies: NUP93 depletion induces formation of actin stress fibers. Combine NUP93 antibodies with stress fiber markers (e.g., LIMCH1) to visualize cytoskeletal reorganization .

• Gene expression analysis: NUP93 modulates genes associated with actin cytoskeleton remodeling and epithelial-to-mesenchymal transition. Correlate NUP93 protein levels (detected by antibodies) with expression of these downstream targets .

• In vivo tumor growth: NUP93 depletion leads to significant defects in tumor establishment and propagation in vivo. NUP93 antibodies can be used for immunohistochemical confirmation of expression levels in tumor xenografts .

• Clinical correlation: High NUP93 and low LIMCH1 expression correlate with late tumor stage in patient samples. Use antibodies for prognostic studies in tumor microarrays .

What are the methodological considerations when studying NUP93 interactions with other nuclear pore complex components?

Investigating NUP93's interactions with other NPC components requires careful experimental design:

• Co-immunoprecipitation: NUP93 forms complexes with NUP53, NUP155, NUP205, and lamin B . Use NUP93 antibodies for co-IP followed by immunoblotting for interacting partners.

• Cellular fractionation: NUP93 localizes to both the basket of the nuclear pore and the nuclear entry of the central gated channel . Use subcellular fractionation followed by Western blotting with NUP93 antibodies to study its distribution.

• Proximity ligation assays: To detect in situ interactions between NUP93 and other nucleoporins in intact cells.

• Effect of mutations: Study how disease-causing mutations (like those in nephrotic syndrome) affect NUP93's ability to interact with other NPC components .

• LINC complex connections: Investigate relationships between NUP93 and the linker of nucleoskeleton and cytoskeleton (LINC) complex, as NUP93 depletion specifically induces significant increases in cytoplasmic Nesprin-1 (307.3% ± 76.3% versus 100.0% ± 18.8% in controls) .

What are common technical issues when using NUP93 antibodies and how can they be resolved?

For Western blot applications specifically, using agarose beads conjugated with anti-FLAG peptide M2 antibody can help enhance specificity when studying tagged versions of NUP93 .

How can I optimize immunofluorescence protocols when studying NUP93 in different cellular contexts?

When performing immunofluorescence for NUP93:

• Fixation optimization:

  • For nuclear membrane proteins, 4% paraformaldehyde fixation typically preserves structure better than methanol

  • If studying NUP93 in relation to actin cytoskeleton (as in cancer studies), avoid methanol fixation which can disrupt actin filaments

• Double-immunofluorescence strategies:

  • For kidney tissues: Combine NUP93 with CD2AP to identify podocytes

  • For endothelial studies: Pair with CD31 marker

  • For cancer research: Combine with markers of epithelial-mesenchymal transition

• Signal interpretation:

  • Normal NUP93 signal appears as dots in nuclei

  • In pathological conditions (mutations), expect diffuse decrease in signal intensity

  • When studying cancer, correlate subcellular localization with invasive phenotype

• Counterstaining recommendations:

  • Include DAPI for nuclear visualization

  • Consider phalloidin staining when studying NUP93's effects on actin cytoskeleton

How do I interpret conflicting data regarding NUP93 function in different cellular contexts?

When facing seemingly contradictory results:

• Cell-type specific effects: NUP93 plays different roles in different tissues. In triple-negative breast cancer, high expression correlates with poor patient survival , while in kidney podocytes, mutations lead to disease .

• Context-dependent mechanisms: In cancer cells, NUP93 modulates genes associated with actin cytoskeleton and EMT , whereas in kidney development, it regulates podocyte migration and proliferation through SMAD4 signaling .

• Dual functionality: Consider NUP93's structural role in the NPC versus its gene regulatory functions. Some experimental approaches may detect one function but miss others.

• Experimental approach differences: Transient versus stable knockdown, antibodies targeting different epitopes, or varying detection methods can all lead to apparently conflicting results.

• Interaction variations: NUP93's interactions with other proteins (NUP53, NUP155, NUP205, lamin B) may vary by cell type , affecting experimental outcomes.

What are the latest methodological advances in studying NUP93's role in nuclear-cytoplasmic communication?

Recent methodological approaches include:

• High-resolution microscopy: Super-resolution techniques to visualize NUP93 within the complex architecture of the NPC.

• Live-cell imaging: Using fluorescently-tagged NUP93 to study dynamics during cell cycle progression and disease states.

• Proteomics approaches: Mass spectrometry-based methods to comprehensively identify NUP93 interaction partners under different conditions.

• CRISPR-based approaches: Gene editing to introduce disease-specific mutations or fluorescent tags at endogenous loci.

• 3D cell culture models: As demonstrated in invasion assays with breast cancer cells, 3D models better recapitulate in vivo NUP93 functions compared to traditional 2D cultures .

• Single-cell analyses: Examining cell-to-cell variation in NUP93 expression and function within heterogeneous populations.