NUP205 Antibody

What is NUP205 and why is it important in cellular research?

NUP205 is a 228 kDa nucleoporin that forms part of the inner ring of the nuclear pore complex (NPC). It plays a crucial role in nuclear pore complex assembly and maintenance, acting as a gatekeeper for nucleocytoplasmic transport . The protein works cooperatively with other nucleoporins to form the NPC scaffold, facilitating the bidirectional movement of proteins and RNA between nuclear and cytoplasmic compartments . NUP205 may specifically anchor NUP62 and other nucleoporins to the NPC, though not NUP153 and TPR . Recent research has expanded our understanding of NUP205 beyond its canonical nuclear pore function, revealing its localization at ciliary bases and its critical role in ciliary formation . Additionally, abnormal NUP205 expression has been implicated in several pathological conditions, including congenital heart disease and lower-grade glioma, making it an important target for diverse research applications .

What are the validated applications for NUP205 antibodies in research?

NUP205 antibodies have been validated for several key research applications. Western blotting (WB) is consistently supported across commercial antibodies, with published results demonstrating detection of the expected 228 kDa band in multiple cell lines including 293T, HeLa, and Jurkat whole cell lysates . Immunoprecipitation (IP) is another validated application, allowing researchers to isolate NUP205 protein complexes to study its interaction partners . While immunofluorescence is less commonly validated for commercial antibodies, researchers have successfully used tagged constructs (such as GFP-NUP205) to visualize NUP205 localization at both nuclear pores and ciliary bases . When selecting a NUP205 antibody, researchers should verify that the specific application has been validated for their species of interest, as some antibodies may work in human samples but have not been confirmed in other organisms despite sequence homology .

How can researchers validate the specificity of NUP205 antibodies?

Validating antibody specificity is critical for reliable NUP205 research. A multi-faceted approach is recommended. First, Western blot analysis should confirm detection at the predicted molecular weight of 228 kDa, as demonstrated with validated antibodies like ab157090 . Second, researchers should include appropriate negative controls, such as analyzing samples where NUP205 has been knocked down via MO (morpholino) or CRISPR-based techniques as implemented in Xenopus models . Third, cross-validation using different antibodies raised against distinct epitopes of NUP205 can strengthen confidence in specificity. For example, comparing results from an antibody targeting the synthetic peptide within Human NUP205 aa 1750-1800 (ab157090) with another targeting a different region . Fourth, rescue experiments provide compelling evidence for specificity, as demonstrated in studies where human NUP205 expression could rescue phenotypes caused by depleting endogenous nup205 . Finally, when studying specific variants, such as the CHD-associated NUP205 p.Pro1610Arg, functional complementation assays can determine whether the antibody detects both wild-type and variant forms with comparable efficiency .

What are the optimal conditions for Western blotting with NUP205 antibodies?

Successful Western blotting for NUP205 requires careful optimization due to its high molecular weight (228 kDa). Based on published protocols, researchers should prepare samples using whole cell lysates rather than fractionated samples to capture the total NUP205 pool . For SDS-PAGE, use low percentage gels (6-7%) or gradient gels to allow proper separation of high molecular weight proteins. Extended running times may be necessary to achieve clear separation. When transferring to membranes, researchers should implement extended transfer times (overnight at low voltage or 2+ hours at higher voltage) using wet transfer systems rather than semi-dry methods to ensure complete transfer of the large protein . Antibody concentrations should be carefully titrated, with successful detection reported using ab157090 at 0.1 μg/mL . For detection, ECL-based systems have proven effective with exposure times around 3 minutes . Researchers should be aware that phosphate-buffered blocking solutions sometimes perform better than milk-based blockers for nuclear pore proteins. Importantly, validation experiments showed clear bands in multiple cell types including 293T, HeLa, and Jurkat cell lysates, making these suitable positive controls when establishing the protocol .

How can researchers effectively use NUP205 antibodies for immunoprecipitation studies?

Immunoprecipitation (IP) with NUP205 antibodies requires optimized protocols due to the protein's involvement in large macromolecular complexes. When preparing lysates, gentler lysis buffers containing 0.5-1% NP-40 or Triton X-100 are recommended to preserve protein-protein interactions. Pre-clearing lysates with protein A/G beads helps reduce non-specific binding. Antibody amounts should be optimized, typically starting with 2-5 μg of antibody per 500 μg of total protein . Researchers should perform overnight incubation at 4°C to maximize antigen-antibody interaction while minimizing degradation. When investigating NUP205 interaction partners, crosslinking approaches may be necessary to capture transient interactions. For co-IP experiments studying NUP205's interaction with other nucleoporins like NUP93, researchers should consider the structural information available regarding their interaction surfaces . For instance, when studying how mutations like p.Pro1610Arg affect interactions, buffer conditions may need adjustment to maintain the integrity of potentially weakened interactions . Sequential IPs can be employed to isolate specific subcomplexes, particularly valuable when distinguishing between NUP205's roles at the nuclear pore versus ciliary bases. Elution can be performed using either acidic conditions or SDS-containing buffers, depending on downstream applications and the stability of the interactions under investigation.

What considerations should be made when using NUP205 antibodies in immunofluorescence studies?

Immunofluorescence (IF) detection of endogenous NUP205 presents certain challenges, as noted in research where suitable antibodies for direct IF detection were unavailable . When direct IF is not possible with available antibodies, researchers have successfully employed tagged constructs such as GFP-NUP205 to visualize localization patterns . This approach revealed that NUP205 localizes not only to the nuclear periphery (expected for a nucleoporin) but also to the base of cilia, suggesting multiple functional roles . For transfection-based approaches using tagged constructs, optimization of expression levels is critical, as overexpression may lead to aggregation or mislocalization. Electroporation has been successfully used for temporal control of expression in developmental models like Xenopus . When studying ciliary localization, co-staining with basal body markers (e.g., γ-tubulin) and ciliary markers (e.g., acetylated tubulin) allows precise determination of NUP205's position relative to these structures . For detecting subcellular mislocalization in disease models, z-stack imaging through the entire cell is recommended, as NUP205 variants like those associated with congenital heart disease may show altered distribution along the apicobasal axis rather than complete absence . Three-dimensional reconstruction imaging is particularly valuable when assessing whether basal bodies with associated NUP205 properly dock at the apical surface, a critical step in normal ciliogenesis that can be disrupted in pathological conditions .

How can NUP205 antibodies be used to investigate ciliopathies and congenital heart defects?

NUP205 antibodies serve as powerful tools for investigating ciliopathies and congenital heart defects, particularly given the recent evidence linking NUP205 to cilia formation and left-right patterning during embryonic development . For ciliopathy research, researchers should implement multi-channel immunofluorescence protocols combining NUP205 antibodies (or GFP-NUP205 constructs) with markers for basal bodies (γ-tubulin) and ciliary axonemes (acetylated tubulin) . This approach allows quantification of multiple parameters including ciliary length, number, and the proper docking of basal bodies at the apical membrane . For congenital heart defect studies, the established link between aberrant left-right patterning and NUP205 dysfunction provides a mechanistic framework for investigation . Researchers can analyze left-right organizer (LRO) cilia in model organisms like Xenopus, where morpholino knockdown of nup205 resulted in abnormal heart looping in 21% of embryos . When studying patient-derived variants, functional complementation assays are particularly valuable, as demonstrated in studies where the NUP205 p.Pro1610Arg variant associated with congenital heart disease failed to rescue the cilia loss phenotype in nup205 morphants, while the p.Thr1044Met variant retained functional capacity . For mechanistic studies, co-immunoprecipitation experiments with NUP205 antibodies can help determine how disease-associated variants affect interactions with partners like NUP93, potentially explaining pathogenic mechanisms through altered protein-protein interactions .

How can researchers utilize NUP205 antibodies to study its role in cancer, particularly lower-grade glioma?

Recent research has revealed a significant association between abnormally high NUP205 expression and lower-grade glioma (LGG) pathogenesis, opening new avenues for cancer research applications of NUP205 antibodies . For translational cancer studies, researchers should implement immunohistochemistry protocols with NUP205 antibodies to assess expression levels in patient tumor samples compared to normal brain tissue, as multiple methods have consistently shown higher NUP205 expression in LGG tumor tissue . When correlating expression with clinical outcomes, a multi-layered approach combining tissue microarrays, Western blotting quantification, and transcriptomic data analysis provides robust evidence, as demonstrated in studies showing that high NUP205 expression serves as an independent risk indicator associated with reduced survival in LGG patients . For mechanistic investigations, researchers can employ NUP205 antibodies in ChIP-seq or RIP-seq approaches to explore the regulatory networks controlled by NUP205, building on GSEA analysis findings that implicate NUP205 in regulating cell cycle progression, Notch signaling, and aminoacyl-tRNA biosynthesis in LGG . To investigate NUP205's potential as an immunotherapeutic target, researchers should design co-immunoprecipitation studies to explore its interaction with immune checkpoint proteins, supported by data showing positive correlations between NUP205 expression and multiple immune checkpoints, particularly PD-L1 . Tumor immune microenvironment studies can combine NUP205 antibodies with markers for specific immune cell populations, especially M2 macrophages, which show strong positive correlation with NUP205 expression in LGG .

What methodologies can be used to investigate the functional redundancy between NUP205 and NUP188?

Research has revealed striking functional redundancy between NUP205 and NUP188 in ciliogenesis, presenting intriguing opportunities for advanced experimental designs . To investigate this redundancy, researchers should implement combinatorial knockdown/knockout approaches targeting both NUP205 and NUP188 simultaneously, as studies in Xenopus demonstrated that co-depletion of both proteins resulted in more severe ciliary phenotypes than individual depletions . Functional complementation experiments provide particularly valuable insights: express human NUP205 in NUP188-depleted cells and vice versa to test interchangeability, following protocols where GFP-NUP205 successfully rescued cilia in nup188 morphants and GFP-NUP188 rescued cilia in nup205 morphants . Structural biology approaches combining antibodies for immunoprecipitation with mass spectrometry can help determine whether NUP205 and NUP188 compete for the same binding partners (especially NUP93) or form mutually exclusive complexes . For temporal control of experimentation, electroporation-based expression of complementing proteins at specific developmental stages has proven effective, allowing researchers to target proteins to ciliary bases rather than nuclear pores based on their differential turnover rates . When investigating functional differences despite redundancy, high-resolution microscopy combined with immunostaining can reveal subtle differences in localization patterns or protein dynamics. Finally, researchers can employ domain-swapping experiments between NUP205 and NUP188 followed by functional assays to identify which regions confer their shared functionality and which regions may be responsible for any non-redundant functions .

What are common challenges when working with NUP205 antibodies and how can they be addressed?

Working with NUP205 antibodies presents several technical challenges that researchers should anticipate. First, the high molecular weight of NUP205 (228 kDa) complicates Western blotting detection. To address this, use low percentage gels (6-7%), extend running times, and implement wet transfer systems with extended transfer duration . Second, non-specific bands may appear, particularly in the 150-200 kDa range. These can be minimized by careful titration of antibody concentration (successful detection has been reported with as little as 0.1 μg/mL for some antibodies) and through the use of gradient gels that provide better resolution of high molecular weight proteins . Third, when studying specific cellular compartments, the dual localization of NUP205 at both nuclear pores and ciliary bases can complicate interpretation . This can be addressed through careful subcellular fractionation protocols or high-resolution microscopy with co-localization markers. Fourth, when studying patient-derived variants, be aware that certain mutations (like p.Pro1610Arg) may affect antibody recognition depending on the epitope location . Using antibodies targeting different epitopes can help ensure detection of variant forms. Fifth, when investigating functional redundancy between NUP205 and NUP188, compensatory mechanisms may mask phenotypes in single knockdown experiments . This can be addressed through simultaneous depletion approaches and careful timing of intervention, as demonstrated in studies using electroporation for temporal control of rescue experiments .

How should researchers approach quantitative analysis of NUP205 expression and localization?

Quantitative analysis of NUP205 expression and localization requires rigorous methodological approaches to ensure accuracy and reproducibility. For Western blot quantification, researchers should use internal loading controls appropriate for nuclear proteins (e.g., lamin B1 rather than cytoplasmic housekeeping proteins) and implement densitometry across multiple biological replicates . Given the evidence that NUP205 expression varies across cancer types and correlates with clinical outcomes in lower-grade glioma, researchers should establish clear thresholds for categorizing "high" versus "low" expression, ideally based on statistical approaches such as median split or optimal cut-point determination as implemented in survival analyses . For localization studies, particularly those investigating NUP205's dual presence at nuclear pores and ciliary bases, automated image analysis workflows can quantify co-localization coefficients with specific markers . When comparing wild-type versus variant forms (such as the CHD-associated p.Pro1610Arg), researchers should implement blinded analysis to prevent bias . For functional quantification, ciliary rescue experiments provide a powerful readout, measuring parameters such as the percentage of multiciliated cells displaying cilia or the average number of cilia per cell after intervention . In the context of tumor immunology studies, bioinformatic approaches correlating NUP205 expression with immune cell infiltration require careful normalization and statistical correction for multiple comparisons, as demonstrated in analyses showing significant correlations between NUP205 expression and multiple immune cell populations, particularly M2 macrophages .

What considerations should be made when designing experiments to test NUP205 variants associated with human disease?

Designing experiments to test disease-associated NUP205 variants requires careful consideration of multiple factors. First, choose appropriate model systems: Xenopus embryos have proven valuable for studying cilia-related functions of NUP205, while patient-derived cells or CRISPR-edited cell lines may be more suitable for studying tissue-specific effects . Second, ensure proper controls: when testing specific variants like p.Pro1610Arg and p.Thr1044Met associated with congenital heart disease, always include wild-type NUP205 rescue conditions for comparison . Third, consider structure-function relationships: the p.Pro1610Arg variant, which failed to rescue cilia loss in nup205 morphants, introduces changes near the NUP93 interaction surface that may increase steric hindrance and alter local protein polarity . Fourth, implement multiple functional readouts: beyond localization, assess the variant's ability to rescue phenotypes (e.g., cilia formation), interact with known partners (e.g., NUP93), and influence downstream signaling pathways . Fifth, for cancer-related studies, integrate data on somatic copy number alterations (SCNA) of NUP205 with immune infiltrate analysis, as demonstrated in research linking NUP205 status to immune cell populations in tumors . Sixth, when studying NUP205 in lower-grade glioma, combine expression analysis with methylation studies to explore epigenetic regulation mechanisms potentially driving overexpression . Finally, for potential therapeutic targeting, carefully assess the functional redundancy between NUP205 and NUP188, as their interchangeability demonstrated in rescue experiments suggests that targeting both proteins may be necessary for complete pathway inhibition .