Phospho-NPM1 (Thr199) Antibody
Description
Overview of Phospho-NPM1 (Thr199) Antibody
Phospho-NPM1 (Thr199) antibodies are recombinant monoclonal or polyclonal antibodies raised against synthetic phosphopeptides corresponding to residues surrounding Thr199 of human NPM1. These antibodies selectively recognize NPM1 phosphorylated at Thr199, a site targeted by CDK2/cyclin E during the G1 phase of the cell cycle .
Key Validation Studies
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Specificity:
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Sensitivity:
| Method | Detection Limit | Advantages |
|---|---|---|
| HTRF | 110 cells/well | No electrophoresis, high-throughput |
| Western Blot | 1,750 cells/well | Traditional validation, requires gels |
Mechanistic Insights
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Cell Cycle Regulation:
Phosphorylation at Thr199 by CDK2/cyclin E triggers NPM1 dissociation from centrosomes, enabling centrosome duplication and mitotic progression . -
Cancer Biology:
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Prostate Cancer: Overexpression of phospho-NPM1 (Thr199) correlates with castration-resistant PCa (CRPC). In CRPC tissues, phospho-NPM1 localized to the nucleoplasm, unlike androgen-dependent PCa (ADPC), where it was nucleolar .
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AML: NPM1 mutations (NPM1c) disrupting its phosphorylation are linked to leukemogenesis .
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Therapeutic Targeting
Product Specs
Target Background
- NPM1 and SURF6 form heterotypic liquid-like droplets in the nucleolus. PMID: 29483575
- High NPM1 expression is associated with tongue neoplasms. PMID: 29746960
- This meta-analysis indicated that NPM may serve as a valuable prognostic biomarker and a potential therapeutic target in human solid tumors. PMID: 30126359
- This paper demonstrates that viral nucleocapsid interacts with NPM1 and protects it from proteolytic cleavage, enhancing cell survival and contributing to porcine epidemic diarrhea virus growth. PMID: 28045037
- DNMT3A R882 mutation plays a significant role in CN-AML patients' prognosis and clinical outcomes both in the presence and absence of NPM1 and FLT3 mutations. PMID: 29079128
- Mutation in the NPM1 gene is associated with Acute Myeloid Leukemia. PMID: 29530994
- Nucleoplasmic translocation of NPM1 is a prerequisite for stress-induced activation of p53. PMID: 27886181
- NPM1 gene B type mutation enhanced the proliferation and invasion of THP-1 AML cells through the regulation of TIMP-2, MMP-2, Ang-1, c-myc and CCND1. PMID: 29441887
- In this study, FLT3 and NPM1 mutations were evaluated in adult Iranian patients with de novo cytogenetically normal acute myeloid leukemia, and their correlations with clinical and laboratory parameters were assessed. PMID: 28294102
- These observations demonstrated that the expression and localization of NPM affected the homeostatic balance of oxidative stress in tumor cells via PRDX6 protein. The regulation axis of NPM/PRDX/ROS may provide a novel therapeutic target for cancer treatment. PMID: 28513872
- These results enhance our understanding of the molecular mechanisms governing nucleoli formation by demonstrating that PPM1D regulates nucleolar formation by modulating NPM phosphorylation status through a novel signaling pathway, PPM1D-CDC25C-CDK1-PLK1. PMID: 27619510
- Data suggest that the direct interaction of several regions of nucleophosmin 1 (NPM1) C-terminal domain (CTD) with cellular membranes could be implicated in diseases where NPM1 is mutated and/or where its overexpression is cytotoxic. PMID: 29330024
- Mechanistically, mutant NPM1 interacted with PML and mediated its delocalization as well as stabilization, contributing to elevated autophagic activity and leukemic cell survival in vitro. PMID: 28740552
- Mutation analysis in NPM1 in acute myeloid leukemia. PMID: 27071442
- We conclude that the degradation of NPM1 and HEXIM1 through autophagy in certain AML subsets contributes to the activation of the BET pathway in these cells. PMID: 27732946
- miR-10b exerts its effects by repressing the translation of KLF4, and NPM1-mA inhibits myeloid differentiation through the miR-10b/KLF4 axis. PMID: 27669739
- NPM1 may play a crucial role in tumor progression in salivary gland adenoid cystic carcinoma (SACC) and is a potential biomarker for SACC. PMID: 27501253
- Data show that phosphorylated forms of nucleophosmin 1 (NPM1) interact with androgen receptor (AR) in the nucleoplasm. PMID: 26993766
- Studies indicate that nucleophosmin 1 (NPM1) has been considered a promising target for the treatment of both hematologic and solid malignancies. PMID: 27058426
- This work identifies the TP53 tumor suppressor as a novel target through which NPM1-RARA impacts leukemogenesis. PMID: 26754533
- These results suggest that relocation of NPM altered its interactional network and consequently disrupted primary functions, including cell proliferation, adhesion, migration, and invasion. NPM plays a promotional role in cancer. PMID: 28262969
- The consequences of mutations in NPM1 and possible mechanisms through which mutations lead to leukemogenesis are reviewed. PMID: 28111462
- NPM1 mutation, but not RUNX1 mutation or multilineage dysplasia, defines a prognostic subgroup within de novo acute myeloid leukemia lacking recurrent cytogenetic abnormalities. PMID: 28370403
- Nucleophosmin 1 (NPM1) mutations in chronic myelomonocytic leukemia and their prognostic relevance. PMID: 28707414
- Multivariable analyses on time to relapse and OS revealed pre-transplant NPM1 MRD levels >1% as an independent prognostic factor for poor survival after allogeneic HSCT, whereas FLT3-ITD had no impact. Notably, the outcome of patients with pre-transplant NPM1 MRD positivity >1% was as poor as that of patients transplanted with RD. PMID: 27471865
- Our results indicate that CD4 expression and older age are adverse prognostic factors in wild-type NPM1, FLT3-ITD-negative CN-AML. PMID: 28318150
- NPM1-dependent nucleolar PIDDosome is a key initiator of the caspase-2 activation cascade. PMID: 28432080
- Data indicate that NPM-ALK was distributed in equal amounts between the cytoplasm and the nucleus. PMID: 26657151
- The aim of this review is to examine the less well-described role of NPM1 in the DNA repair pathways, as well as its role in the regulation of apoptosis and its mutation in cancers. [review] PMID: 27553022
- This study revealed that the localization of fluorescently labeled NPM is affected by the interaction between various forms of the protein. PMID: 28384310
- The value of mutated NPM1 in AML in risk assessment and evaluating prognosis. PMID: 27416910
- NPM1 downregulation by P-STAT5 is mediated by impairing the BRCA1-BARD1 ubiquitin ligase, which controls the stability of NPM1. In turn, decreased NPM1 levels led to suppression of p53 expression, resulting in enhanced cell survival. PMID: 28005077
- These results suggest that the p38/NPM/PP2A complex acts as a dynamic sensor, allowing endothelial cells to react rapidly to acute oxidative stress. PMID: 27142525
- Nucleophosmin-anaplastic lymphoma kinase serves as the founding member of the ALK fusion protein family, and its role in malignant cell transformation is by far the best characterized and, thus, is the main focus of this review. [review] PMID: 27879258
- Data suggest that NPM1 mutations are a secondary or late event in the pathogenesis of AML and are preceded by founder mutations in genes that may be associated with recently described preclinical states such as clonal hematopoiesis of indeterminate potential or clonal cytopenias of undetermined significance. PMID: 28152414
- Mutations of the NPM1 gene are associated with Acute myeloid leukemia. PMID: 27636548
- RQ-PCR of the NPM1 type A mutation was more sensitive and reliable than MFC for determination of minimal residual disease, which might have clinical implications. PMID: 27191933
- The results demonstrated that NPM downregulation markedly reversed the effects of multidrug resistance in human hepatoma cells. Additionally, NPM downregulation reduced P-glycoprotein expression, as well as MDR1 expression. PMID: 28259962
- Only karyotype and mutated NPM1 (NPM1mut) were independent predictors of survival in acute myeloid leukemia. PMID: 27643573
- Our study provides a method for systematic characterization of NPM1 oligomer formation changes and for screening inhibitors of NPM1 oligomerization. PMID: 27983985
- Mutation of NPM1 determined by the widely available and inexpensive Immunohistochemical (IHC) agrees closely with results of the standard molecular methods. Thus, technically and financially not well-endowed laboratories can provide the prognostically and potentially therapeutically important information on NPM1 mutation using IHC. PMID: 27748301
- In pediatric patients with AML from Argentina, a favorable prognosis of AML with genotype NPM1-mutated/FLT3-ITD-negative was confirmed. PMID: 27436336
- This study investigated the expression level of miR-1, miR-486, and let-7a in 45 CN-AML patients well characterized for FLT3 and/or NPM1 mutations using real-time quantitative RT-PCR and evaluated the association between candidate miRs expression and clinical features. PMID: 26526573
- Data showed that the pre-transplant level of MRD in patients with normal karyotype AML harboring NPM1 mutation in CR provides important prognostic information, which, as an independent prognostic factor, predicts transplant results. PMID: 27798920
- Molecular subtypes of NPM1 mutations have different clinical profiles, specific patterns of accompanying molecular mutations, and varying outcomes in intermediate risk acute myeloid leukemia. PMID: 26471486
- In this study, a direct association was observed between NPMc(+)expression in AML, reduced antioxidant responses, and enhanced sensitivity to an oral proteasome inhibitor that induces oxidative stress. PMID: 26634271
- Analysis of the frequency and features of Acute myeloid leukemia with mutated NPM1 in Indian patients. PMID: 26669619
- Nucleophosmin Interacts with promyelocytic leukemia protein/retinoic acid receptor alpha Only in the Resistant Cell Line. PMID: 26997274
- Longitudinal qPCR monitoring of nucleophosmin 1 mutations after allogeneic hematopoietic stem cell transplantation to predict AML relapse. PMID: 26642331
- Results suggest that cup-like nuclei represent an important morphologic clue that can predict NPMc+ AML and guide toward prioritizing the further workup of AML patients. PMID: 26200838
Q&A
What is NPM1 and why is the phosphorylation at Thr199 significant?
NPM1 (Nucleophosmin 1, also known as B23, nutramin, or NO38) is a ubiquitously expressed phosphoprotein involved in multiple cellular processes including ribosome assembly/transport, cytoplasmic/nuclear trafficking, regulation of DNA polymerase alpha activity, centrosome duplication, and regulation of p53. Phosphorylation at Thr199 is particularly significant because it is mediated by CDK2-cyclin E during cell cycle progression and is a prerequisite for centrosome duplication. Upon phosphorylation at Thr199, NPM1 dissociates from centrosomes, allowing them to initiate duplication . This specific phosphorylation is therefore a critical regulatory mechanism in cell division and proliferation.
What are the cellular localization patterns of phosphorylated NPM1 (Thr199)?
Phosphorylated NPM1 (Thr199) displays distinct subcellular localization patterns that differ from total NPM1. While total NPM1 is predominantly found in the nucleolus, phospho-NPM1 (Thr199) is localized in sub-nuclear structures known as nuclear speckles in the nucleoplasm . These speckles can be identified as dense regions in bright field microscopy and are positive for the sc-35 marker . Additionally, phospho-NPM1 (Thr199) expression increases during mitosis, peaking at metaphase and decreasing from anaphase to interphase . This dynamic localization pattern is crucial for understanding its functional role in cell cycle regulation.
What is the molecular weight of phosphorylated NPM1?
The calculated molecular weight of NPM1 is approximately 32-33 kDa , but in Western blot analysis, phosphorylated NPM1 (Thr199) typically appears as a band at approximately 38 kDa . This difference between theoretical and observed molecular weight may be attributed to post-translational modifications, particularly phosphorylation, which can affect protein migration in SDS-PAGE.
What are the validated applications for phospho-NPM1 (Thr199) antibodies?
Phospho-NPM1 (Thr199) antibodies have been primarily validated for Western blotting (WB) with typical working dilutions ranging from 1:500 to 1:2000 . Some antibodies have also been validated for immunohistochemistry (IHC) on formalin/PFA-fixed paraffin-embedded tissue sections . When using these antibodies for Western blotting, researchers should optimize the dilution depending on their specific experimental conditions and the antibody source. For IHC applications, antigen retrieval methods such as high-pressure and temperature sodium citrate (pH 6.0) treatment have been reported to be effective .
How can phospho-NPM1 (Thr199) antibodies be used to study centrosome duplication?
To study centrosome duplication using phospho-NPM1 (Thr199) antibodies, researchers can employ multiple complementary approaches:
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Co-immunofluorescence microscopy: Stain cells with phospho-NPM1 (Thr199) antibody along with centrosome markers (e.g., γ-tubulin) to visualize their association and dissociation during cell cycle .
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Cell synchronization: Synchronize cells at different cell cycle stages and analyze phospho-NPM1 (Thr199) levels by Western blot to correlate with centrosome duplication events .
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CDK2 inhibition studies: Treat cells with specific CDK2 inhibitors and assess the impact on Thr199 phosphorylation and centrosome duplication .
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Live-cell imaging: Use fluorescently tagged NPM1 along with phospho-specific antibodies in fixed-time points to track the dynamics of phosphorylation during centrosome duplication .
This multi-faceted approach allows researchers to establish causative relationships between NPM1 phosphorylation and centrosome duplication events.
What controls should be included when using phospho-NPM1 (Thr199) antibodies?
For rigorous experimental design with phospho-NPM1 (Thr199) antibodies, the following controls should be included:
These controls ensure the reliability and interpretability of results obtained with phospho-NPM1 (Thr199) antibodies.
How can phospho-NPM1 (Thr199) be used as a biomarker in cancer research?
Phospho-NPM1 (Thr199) shows potential as a biomarker in cancer research, particularly in prostate cancer progression. Research has shown that both total NPM1 and its phosphorylated forms (Thr199 and Thr234/237) are significantly overexpressed in castration-resistant prostate cancer (CRPC) compared to androgen-dependent prostate cancer (ADPC) . Implementing phospho-NPM1 (Thr199) as a cancer biomarker requires:
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Tissue microarray analysis: Comparing phospho-NPM1 (Thr199) expression across cancer stages and grades .
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Correlation studies: Analyzing relationships between phospho-NPM1 (Thr199) levels and clinical parameters such as Gleason score, PSA levels, and disease recurrence .
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Multiplex immunohistochemistry: Combining phospho-NPM1 (Thr199) with other markers to create more robust predictive panels.
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Outcome association: Correlating phospho-NPM1 (Thr199) expression with patient survival and treatment response.
While current research indicates its potential utility, further large-scale validation studies are needed to establish phospho-NPM1 (Thr199) as a clinically relevant biomarker.
What is the relationship between phospho-NPM1 (Thr199) and androgen receptor signaling in prostate cancer?
Phosphorylated forms of NPM1, including at Thr199, interact with androgen receptor (AR) in the nucleoplasm, suggesting a functional relationship in prostate cancer progression . Research demonstrates that:
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While total NPM1 is primarily localized in the nucleolus and AR in the nucleoplasm, their phosphorylated forms colocalize in nuclear speckles .
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Co-immunoprecipitation studies confirm that phospho-NPM1 (Thr199) interacts with AR .
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Inhibition of NPM1 phosphorylation (e.g., by the pseudopeptide N6L) correlates with reduced AR activity .
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Both phosphorylated NPM1 forms and AR are overexpressed in castration-resistant prostate cancer .
These findings suggest that phospho-NPM1 (Thr199) may contribute to prostate cancer progression through modulation of AR signaling, particularly in the development of castration resistance. Researchers investigating this relationship should combine biochemical approaches with functional studies to elucidate the precise mechanisms involved.
How can phospho-NPM1 (Thr199) antibodies be used to evaluate potential anti-cancer therapeutics?
Phospho-NPM1 (Thr199) antibodies can serve as valuable tools in evaluating potential anti-cancer therapeutics, particularly those targeting cell cycle regulation or NPM1 function. Methodological approaches include:
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Drug screening: Use Western blotting with phospho-NPM1 (Thr199) antibodies to identify compounds that reduce NPM1 phosphorylation, such as the pseudopeptide N6L .
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Dose-response studies: Quantify the relationship between drug concentration and reduction in phospho-NPM1 (Thr199) levels .
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Time-course analysis: Monitor changes in phospho-NPM1 (Thr199) levels following drug treatment to determine optimal treatment duration .
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Combination therapy assessment: Evaluate how standard-of-care treatments (e.g., hormonotherapy, docetaxel) affect phospho-NPM1 (Thr199) levels when used alone or in combination with novel therapeutics .
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Correlation with functional outcomes: Link changes in phospho-NPM1 (Thr199) to functional endpoints such as cell proliferation, apoptosis, and tumor growth in xenograft models .
This approach has been successfully employed with N6L, which inhibits NPM1 phosphorylation and demonstrates anti-tumor activity in prostate cancer models .
How can researchers address non-specific binding when using phospho-NPM1 (Thr199) antibodies?
Non-specific binding can compromise the reliability of results obtained with phospho-NPM1 (Thr199) antibodies. To address this common challenge:
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Optimize blocking conditions: Test different blocking agents (BSA, non-fat dry milk, commercial blockers) at various concentrations and incubation times.
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Adjust antibody dilution: Perform titration experiments to determine the optimal antibody concentration that maximizes specific signal while minimizing background.
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Modify washing protocols: Increase the number, duration, or stringency of washes (by adding detergents like Tween-20) between antibody incubations.
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Use phospho-blocking agents: Include phosphatase inhibitors in sample preparation to preserve phosphorylation status and reduce background.
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Consider alternative antibody formats: If polyclonal antibodies show high background, try monoclonal alternatives that may offer greater specificity .
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Validate with competing peptides: Use both phosphorylated and non-phosphorylated peptides corresponding to the Thr199 region to confirm signal specificity.
These methodological adjustments can significantly improve the signal-to-noise ratio and enhance data reliability.
How should researchers interpret changes in phospho-NPM1 (Thr199) levels during cell cycle progression?
Interpreting changes in phospho-NPM1 (Thr199) levels during cell cycle progression requires careful consideration of several factors:
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Baseline reference: Establish baseline phospho-NPM1 (Thr199) levels in quiescent (G0) cells as a reference point.
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Cell synchronization validation: Confirm cell cycle synchronization using established markers (e.g., cyclin expression, DNA content) before interpreting phospho-NPM1 (Thr199) changes.
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Temporal dynamics: Recognize that phospho-NPM1 (Thr199) levels typically increase during mitosis, peaking at metaphase and decreasing from anaphase to interphase .
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Spatial considerations: Note that phospho-NPM1 (Thr199) relocalizes from centrosomes to nuclear speckles during cell cycle progression .
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Quantitative analysis: Use densitometry for Western blots or fluorescence intensity measurements for immunofluorescence to quantify changes objectively.
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Normalization approach: When quantifying, normalize phospho-NPM1 (Thr199) to total NPM1 rather than housekeeping proteins to account for changes in total protein levels.
Understanding these dynamics allows researchers to correctly interpret fluctuations in phospho-NPM1 (Thr199) levels and their biological significance.
What factors can lead to false negative or false positive results when using phospho-NPM1 (Thr199) antibodies?
Several factors can compromise the accuracy of phospho-NPM1 (Thr199) antibody results:
| Factor Type | False Negative Causes | False Positive Causes |
|---|---|---|
| Sample preparation | - Insufficient phosphatase inhibitors - Excessive sample heating - Prolonged storage without protease inhibitors | - Cross-reactivity with similar phosphorylation sites - Denaturation artifacts |
| Technical factors | - Suboptimal antibody dilution - Inadequate antigen retrieval - Poor transfer efficiency in Western blotting | - Excessive antibody concentration - Insufficient blocking - Inadequate washing |
| Biological variables | - Cell cycle heterogeneity - Low baseline phosphorylation - Competing phosphorylation at nearby residues | - Stress-induced phosphorylation during sample handling - Non-specific CDK2 activation |
| Reagent quality | - Antibody degradation - Loss of specificity over time - Batch-to-batch variation | - Contamination - Cross-reactivity evolution in polyclonal sources |
Researchers should systematically address these factors through appropriate controls and methodology optimization to ensure reliable and reproducible results.
How can phospho-NPM1 (Thr199) analysis be integrated into high-throughput screening platforms?
Integrating phospho-NPM1 (Thr199) analysis into high-throughput screening platforms requires adapting traditional techniques to automated, scalable formats:
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ELISA-based detection: Develop sandwich ELISAs using capture antibodies against total NPM1 and detection antibodies against phospho-NPM1 (Thr199).
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Automated Western blotting: Implement capillary-based or microfluidic Western systems that require less sample and enable higher throughput.
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High-content imaging: Utilize automated microscopy systems with phospho-NPM1 (Thr199) antibodies to simultaneously analyze phosphorylation status and subcellular localization.
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Phospho-flow cytometry: Adapt phospho-NPM1 (Thr199) antibodies for flow cytometry to rapidly analyze large cell populations.
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Reverse phase protein arrays (RPPA): Spot cellular lysates onto microarrays and probe with phospho-NPM1 (Thr199) antibodies for parallel analysis of multiple samples.
These approaches enable screening compound libraries for modulators of NPM1 phosphorylation, potentially identifying novel anti-cancer therapeutics targeting cell cycle regulation.
What are the emerging techniques for studying the dynamics of NPM1 Thr199 phosphorylation in live cells?
Emerging techniques for studying phospho-NPM1 (Thr199) dynamics in live cells offer temporal and spatial resolution previously unattainable:
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Phospho-specific fluorescent biosensors: Develop FRET-based sensors that change conformation upon Thr199 phosphorylation, enabling real-time visualization.
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Antibody fragment-based imaging: Use cell-permeable phospho-specific single-chain antibody fragments conjugated to fluorescent proteins.
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SunTag amplification system: Employ the SunTag system combined with phospho-specific nanobodies for amplified fluorescent signal detection.
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Proximity ligation assays (PLA): Adapt PLA for temporal analysis of phospho-NPM1 (Thr199) interactions with binding partners like AR.
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CRISPR-based endogenous tagging: Generate cell lines with fluorescently tagged endogenous NPM1 combined with phospho-specific detection methods.
These approaches overcome limitations of traditional fixed-cell immunofluorescence, allowing researchers to monitor phosphorylation dynamics during processes like centrosome duplication and mitosis with unprecedented resolution.
How might single-cell analysis of phospho-NPM1 (Thr199) advance our understanding of cancer heterogeneity?
Single-cell analysis of phospho-NPM1 (Thr199) offers transformative potential for understanding cancer heterogeneity:
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Single-cell Western blotting: Analyze phospho-NPM1 (Thr199) levels in individual cells to reveal heterogeneity masked by bulk analysis.
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Mass cytometry (CyTOF): Combine phospho-NPM1 (Thr199) antibodies with metal-tagged antibodies against other cancer markers for high-dimensional profiling.
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Single-cell imaging mass spectrometry: Visualize the spatial distribution of phospho-NPM1 (Thr199) within tissue sections at single-cell resolution.
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Digital spatial profiling: Quantify phospho-NPM1 (Thr199) levels in spatially resolved single cells within the tumor microenvironment.
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Integrated multi-omics: Correlate single-cell phospho-NPM1 (Thr199) levels with transcriptomic or genomic profiles from the same cells.
