NAT10 Antibody, FITC conjugated

What is NAT10 and why is it significant in cancer research?

NAT10 (N-acetyltransferase 10) is a nucleolar protein of approximately 116 kDa that plays crucial roles in multiple cellular processes including regulation of telomerase activity, DNA damage response, and cytokinesis. It is particularly significant in cancer research because it functions as the enzyme responsible for N4-acetylcytidine (ac4C) modification of mRNA, a process that ensures RNA stability and effective translation .

Recent studies have demonstrated that elevated NAT10 expression correlates with poor prognosis in various cancers including nasopharyngeal carcinoma (NPC) and breast cancer. The NAT10/ac4C/DDX5 axis has been shown to upregulate high mobility group box 1 (HMGB1) and inhibit CD4+ and CD8+ T cells, contributing to immunosuppression in cancer microenvironments . Additionally, NAT10 mediates ac4C modification of multidrug resistance proteins like MDR1 and BCRP, potentially contributing to chemoresistance mechanisms .

What are the most common applications of fluorescently conjugated NAT10 antibodies?

Fluorescently conjugated NAT10 antibodies are primarily used in the following research applications:

• Immunofluorescence (IF)/Immunocytochemistry (ICC): For visualizing NAT10 localization in cells, typically at dilutions of 1:200-1:800 for unconjugated antibodies and 1:400-1:1600 for directly conjugated antibodies .

• Flow Cytometry (intracellular): For quantifying NAT10 expression at the single-cell level, typically using 0.40 μg per 10^6 cells in a 100 μl suspension .

• Protein co-localization studies: For examining spatial relationships between NAT10 and potential interacting partners or subcellular compartments.

• Live-cell imaging: For tracking dynamics of NAT10 in real-time when using cell-permeable fluorescent conjugates.

The fluorescently conjugated antibodies eliminate the need for secondary antibodies, reducing background and simplifying multiplexing experiments when studying NAT10 alongside other proteins of interest .

What is the difference between CoraLite 488 and FITC conjugated NAT10 antibodies?

While both fluorophores emit in the green spectrum, there are several important distinctions researchers should consider:

CoraLite 488 conjugated NAT10 antibodies (such as CL488-82585) offer improved photostability compared to traditional FITC conjugates, making them more suitable for extended imaging sessions and confocal microscopy applications . The excitation/emission properties (493 nm/522 nm) are compatible with standard FITC filter sets, allowing researchers to use existing equipment without modification . When designing experiments requiring multiple fluorescent markers, these spectral characteristics should be considered to minimize bleed-through with other fluorophores.

How should I optimize immunofluorescence protocols for detecting nuclear NAT10 expression?

Optimizing immunofluorescence for nuclear NAT10 detection requires careful attention to several parameters:

• Fixation method: Use 4% paraformaldehyde for 15 minutes at room temperature to preserve nuclear architecture while maintaining antibody epitope accessibility.

• Permeabilization: Since NAT10 is a nucleolar protein, thorough permeabilization is essential. Use 0.2-0.5% Triton X-100 for 10 minutes to ensure antibody access to nuclear compartments.

• Blocking: Implement a robust blocking step (5% normal serum + 0.3% Triton X-100 in PBS) for at least 1 hour to reduce background signal.

• Antibody dilution: Begin with the manufacturer's recommended dilution range (e.g., 1:400-1:1600 for CoraLite 488 conjugated antibodies) , then optimize through titration experiments.

• Nuclear counterstaining: Include DAPI or Hoechst staining to clearly define nuclear boundaries and facilitate proper assessment of NAT10 nuclear localization.

• Confocal microscopy settings: Use optical sectioning to accurately visualize nucleolar localization, with optimal pinhole settings to reduce out-of-focus light.

• Positive controls: Include HeLa cells, which are known to express detectable levels of NAT10 and have been validated for both antibodies mentioned in the search results .

Remember that the nucleolar localization pattern of NAT10 should be distinct from general nuclear staining, appearing as concentrated foci within the nucleus, particularly in actively proliferating cells.

What are the critical considerations when designing flow cytometry experiments with fluorescently labeled NAT10 antibodies?

When designing flow cytometry experiments using fluorescently labeled NAT10 antibodies, researchers should consider:

• Fixation and permeabilization: Since NAT10 is an intracellular, primarily nucleolar protein, robust permeabilization is required. Use commercially available intracellular staining kits compatible with nuclear proteins.

• Antibody concentration: The recommended starting concentration is 0.40 μg per 10^6 cells in a 100 μl suspension , but titration experiments should be performed to determine optimal signal-to-noise ratio.

• Compensation controls: Include single-color controls if performing multicolor flow cytometry to correct for spectral overlap between fluorophores.

• Negative controls: Include isotype controls (Rabbit IgG conjugated to the same fluorophore) at equivalent concentrations to assess non-specific binding .

• Positive controls: HeLa cells serve as reliable positive controls as they express detectable levels of NAT10 .

• Cell cycle considerations: Since NAT10 may exhibit cell cycle-dependent expression patterns, consider co-staining with DNA content markers to correlate NAT10 expression with cell cycle phases.

• Gating strategy: Implement a sequential gating strategy that first identifies viable single cells before analyzing NAT10 expression to eliminate artifacts from cell aggregates or debris.

• Signal amplification: For detecting subtle changes in NAT10 expression, consider whether signal amplification techniques might be necessary.

How can I validate the specificity of NAT10 antibody staining in my experimental system?

Validating antibody specificity is critical for ensuring reliable experimental results. For NAT10 antibodies, consider these validation approaches:

• RNA interference: Perform siRNA or shRNA knockdown of NAT10 and confirm reduction in antibody signal. This approach has been previously validated for NAT10 antibodies (PMID: 24786082) .

• CRISPR-Cas9 knockout: Generate NAT10 knockout cell lines as negative controls, which should show absence of specific staining.

• Overexpression controls: Transfect cells with NAT10 expression vectors and confirm increased antibody signal intensity.

• Peptide competition: Pre-incubate the antibody with immunizing peptide (if available) before staining to block specific binding sites.

• Multiple antibody validation: Compare staining patterns using antibodies targeting different epitopes of NAT10.

• Western blot correlation: Perform western blot analysis on the same samples used for immunofluorescence to confirm that detected protein is of the expected molecular weight (116 kDa) .

• Subcellular localization: Confirm that the staining pattern is consistent with expected nucleolar localization of NAT10.

• Species reactivity verification: If working with non-human samples, verify cross-reactivity with your species of interest, as the antibodies mentioned have confirmed reactivity with human and mouse samples .

How can I use NAT10 antibodies to investigate its role in RNA acetylation and cancer progression?

To investigate NAT10's role in RNA acetylation and cancer progression, researchers can implement these advanced approaches:

• ac4C-RIP coupled with RNA-seq: Use NAT10 antibodies to immunoprecipitate NAT10 complexes, followed by ac4C-specific antibodies to identify NAT10-mediated ac4C-modified RNA targets, as demonstrated in recent breast cancer studies .

• Dual immunofluorescence: Combine fluorescently conjugated NAT10 antibodies with antibodies against potential downstream targets (e.g., DDX5, HMGB1) to examine co-localization and potential functional relationships .

• ChIP-seq and RIP-seq integration: Combine chromatin immunoprecipitation (ChIP) using transcription factor antibodies with RNA immunoprecipitation (RIP) using NAT10 antibodies to identify relationships between transcriptional regulation and post-transcriptional acetylation .

• Proximity ligation assay (PLA): Use NAT10 antibodies in combination with antibodies against suspected interacting proteins to visualize and quantify protein-protein interactions in situ.

• CRISPR-Cas9 editing with rescue experiments: Knockout NAT10 and perform rescue experiments with wild-type versus enzymatically inactive NAT10 to distinguish between acetylation-dependent and independent functions.

• Patient-derived xenograft (PDX) models: Apply NAT10 antibodies to track protein expression in PDX models treated with NAT10 inhibitors like remodelin to correlate molecular responses with treatment outcomes .

• Tissue microarray analysis: Use immunohistochemistry with NAT10 antibodies on cancer tissue microarrays to correlate expression with patient outcomes across cancer types and stages.

These approaches can help elucidate the mechanistic roles of NAT10 in cancer progression and identify potential therapeutic strategies targeting NAT10-mediated pathways.

What are the considerations for investigating NAT10's role in T-cell immunosuppression using fluorescently conjugated antibodies?

When studying NAT10's role in T-cell immunosuppression, researchers should consider:

• Multi-parameter flow cytometry panels: Design panels combining NAT10 detection with T-cell markers (CD4, CD8), activation markers (CD69, CD25), and functional markers (IFN-γ, IL-2) to correlate NAT10 expression with T-cell function.

• Primary T-cell isolation protocols: Optimize fixation and permeabilization methods specifically for primary T-cells, which may differ from established cell lines. Typically, methanol-based permeabilization provides better nuclear antigen access.

• Tumor-infiltrating lymphocyte (TIL) analysis: When studying tumor samples, include strategies to distinguish TILs from other cells in the tumor microenvironment.

• In vitro T-cell functional assays: Combine NAT10 staining with proliferation assays (CFSE dilution) or cytotoxicity assays to correlate NAT10 expression with functional outcomes.

• Single-cell analysis: Consider incorporating NAT10 antibodies into single-cell RNA-seq or CyTOF panels to correlate protein expression with transcriptional profiles at the single-cell level.

• Checkpoint inhibitor response correlation: As NAT10 inhibition has been shown to increase sensitivity to PD-1 therapy , design experiments to investigate the relationship between NAT10 expression and checkpoint inhibitor efficacy.

• ex vivo T-cell models: Develop ex vivo culture systems where T-cells are exposed to tumor-derived factors to study how microenvironmental cues affect NAT10 expression and function.

Research has demonstrated that the NAT10/ac4C/DDX5 axis upregulates HMGB1 and inhibits CD4+ and CD8+ T cells, suggesting NAT10 may be a target for cancer immunotherapy approaches .

How can I develop a quantitative imaging workflow to measure changes in NAT10 expression and localization after drug treatment?

To develop a robust quantitative imaging workflow for assessing drug-induced changes in NAT10 expression and localization:

• High-content imaging setup: Use automated microscopy platforms with consistent illumination and acquisition parameters to ensure comparable data across multiple conditions.

• Standardized sample preparation: Implement rigorous protocols for cell seeding density, fixation timing, and antibody staining to minimize technical variations.

• Nuclear segmentation pipeline: Develop an image analysis pipeline that accurately segments nuclei (using DAPI) and nucleoli (using fibrillarin or nucleolin co-staining) to create masks for quantifying NAT10 signal.

• Multi-parameter measurements:

  • Total NAT10 intensity per cell

  • Nuclear vs. cytoplasmic distribution ratio

  • Nucleolar enrichment index

  • Number and size of NAT10-positive foci

  • Colocalization coefficients with other markers of interest

• Time-lapse experiments: For live-cell imaging applications, establish photobleaching correction methods and normalized intensity measurements to track dynamic changes in NAT10 localization.

• Statistical analysis workflow: Implement appropriate statistical tests (e.g., ANOVA with post-hoc tests) and visualization methods to analyze distribution patterns across treatment conditions.

• Validation with biochemical assays: Correlate imaging data with western blot quantification to validate expression-level changes detected by microscopy.

• Machine learning approaches: Consider implementing machine learning classifiers to detect subtle phenotypic changes in NAT10 distribution patterns that may not be captured by conventional intensity measurements.

This workflow would be particularly valuable for evaluating the effects of NAT10 inhibitors like remodelin, which has shown promise in overcoming chemoresistance in breast cancer models .

Why might I observe inconsistent staining patterns with NAT10 antibodies, and how can I resolve these issues?

Inconsistent staining patterns with NAT10 antibodies can arise from several sources:

• Cell cycle variation: NAT10 expression and localization may vary throughout the cell cycle. Synchronize cells or co-stain with cell cycle markers to account for this variability.

• Fixation artifacts: Overfixation can mask epitopes while underfixation may compromise cellular architecture. Test different fixation durations (10-20 minutes) with 4% paraformaldehyde and determine optimal conditions for your cell type.

• Insufficient permeabilization: Since NAT10 is a nucleolar protein, inadequate permeabilization may prevent antibody access. Consider testing stronger permeabilization conditions (0.5% Triton X-100 for 15 minutes) or methanol permeabilization for detection of nuclear antigens.

• Antibody degradation: Fluorescently conjugated antibodies may degrade with repeated freeze-thaw cycles or extended storage at improper temperatures. Aliquot antibodies upon receipt and store at -20°C protected from light as recommended in the storage conditions .

• Protocol optimization by cell type: Different cell types may require modified protocols. For cell lines not previously validated (beyond HeLa, A549, NIH/3T3), perform titration experiments to determine optimal antibody concentration.

• Antigen masking: Protein-protein interactions may mask the epitope in certain contexts. Consider different epitope retrieval methods if working with fixed tissues rather than cultured cells.

• Batch variation: If inconsistencies appear between experiments, implement a standardized positive control (e.g., HeLa cells) in each experiment to normalize results across batches.

• Photobleaching during imaging: The CoraLite 488 conjugate offers improved photostability compared to FITC, but still requires careful management of exposure times and illumination intensity . Establish consistent imaging parameters and minimize sample exposure during microscope setup.

What are the best practices for quantifying NAT10 expression levels in heterogeneous tumor samples?

When quantifying NAT10 expression in heterogeneous tumor samples, consider these best practices:

• Multiplex immunofluorescence approach: Combine NAT10 antibodies with markers for different cell types (e.g., cytokeratins for tumor cells, CD45 for immune cells) to accurately identify and quantify NAT10 within specific cell populations.

• Tissue microarray utilization: If analyzing multiple patient samples, tissue microarrays allow for standardized staining conditions across specimens.

• Standardized image acquisition: Establish consistent exposure settings calibrated to control samples to ensure comparable intensity measurements across specimens.

• Digital pathology workflow:

  • Cell segmentation based on nuclear and membrane markers

  • Classification of cells into relevant subtypes

  • Quantification of NAT10 intensity within each cell subpopulation

  • Spatial analysis of NAT10 expression relative to microenvironmental features

• Internal reference standards: Include known positive cell types on each slide as internal calibration standards for intensity normalization.

• Multi-region sampling: For larger tumors, analyze multiple regions to account for intratumoral heterogeneity.

• Correlation with other methodologies: Validate immunofluorescence findings with orthogonal methods like western blotting or qPCR when possible.

• Clinical data integration: Correlate NAT10 expression patterns with patient outcomes, as elevated NAT10 has been associated with poor prognosis in multiple cancer types .

• Automation and machine learning: For large sample sets, implement automated image analysis workflows with machine learning algorithms trained to recognize specific cell types and quantify marker expression.

How should I optimize dual immunofluorescence protocols when combining NAT10 antibodies with other markers?

For optimizing dual immunofluorescence protocols combining NAT10 with other markers:

• Antibody compatibility assessment:

  • Check species compatibility to avoid cross-reactivity between primary or secondary antibodies

  • Verify that fixation and permeabilization conditions are compatible for all target antigens

  • Test antibodies individually before combining to establish baseline staining patterns

• Sequential vs. simultaneous staining strategies:

  • For multiple rabbit-derived antibodies, sequential staining with intermediate blocking steps may be necessary

  • For antibodies from different species, simultaneous incubation often works well

  • If one antigen is significantly less abundant, stain for it first to ensure detection

• Fluorophore selection to minimize bleed-through:

  • When using CoraLite 488 conjugated NAT10 antibody (excitation/emission: 493/522 nm) , pair with fluorophores separated by at least 50 nm in emission spectrum

  • Consider spectral unmixing during image acquisition if using closely overlapping fluorophores

• Antibody concentration optimization:

  • Titrate each antibody individually, then in combination

  • The recommended dilution for fluorescently conjugated NAT10 antibodies (1:400-1:1600) may need adjustment in multiplexed protocols

• Blocking strategy refinement:

  • Implement sequential blocking steps with normal sera from different species

  • Consider using Fab fragment blocking to minimize cross-reactivity in multi-species antibody panels

• Controls for multiplexed staining:

  • Single antibody controls to verify specificity in the multiplex context

  • Secondary-only controls to assess non-specific binding

  • Absorption controls to confirm specificity of each signal

• Image acquisition optimization:

  • Acquire channels sequentially rather than simultaneously to minimize bleed-through

  • Implement careful exposure settings to prevent oversaturation while maintaining sensitivity

• Nuclear landmark co-staining:

  • Include DAPI for nuclear counterstaining

  • Consider additional nucleolar markers (fibrillarin, nucleolin) to precisely define NAT10 localization within subnuclear compartments

How can NAT10 antibodies be used to study resistance to cancer therapies?

NAT10 antibodies can be valuable tools for investigating therapy resistance mechanisms:

• Expression correlation with drug response: Use fluorescently conjugated NAT10 antibodies in flow cytometry to correlate expression levels with drug response profiles across patient samples or cell line panels.

• Drug resistance mechanisms investigation: Recent research has shown that NAT10 mediates ac4C modification of multidrug resistance protein (MDR1) and breast cancer resistance protein (BCRP) mRNAs, enhancing their stability and translation efficiency . Antibodies can help track these relationships in patient samples.

• Remodelin treatment monitoring: The NAT10 inhibitor remodelin has been shown to reinstate susceptibility to capecitabine in resistant breast cancer cells . NAT10 antibodies can be used to monitor NAT10 levels during treatment and correlate with response.

• Checkpoint inhibitor therapy enhancement: Research has demonstrated that inhibition of NAT10 increases sensitivity to PD-1 therapy . Immunofluorescence with NAT10 antibodies can help identify patients who might benefit from combined NAT10 inhibition and checkpoint blockade.

• Patient stratification biomarker development: Develop immunohistochemistry or immunofluorescence protocols using NAT10 antibodies that could be translated to clinical pathology workflows for patient stratification.

• Chemotherapy resistance mechanisms: Investigate the connection between NAT10 expression, ac4C modification, and expression of ABC transporters in patient-derived samples to identify potential resistance mechanisms.

• Combination therapy rationale: Use mechanistic insights from NAT10 antibody-based studies to develop rational combination strategies targeting both NAT10 and resistance-mediating factors.

What experimental approaches can help elucidate the relationship between NAT10 and RNA acetylation in cancer cells?

To investigate NAT10's role in RNA acetylation in cancer:

• ac4C-RIP-seq: Combine RNA immunoprecipitation using ac4C-specific antibodies with sequencing to identify ac4C-modified transcripts in cancer cells with varying NAT10 expression levels .

• NAT10 knockdown/overexpression with ac4C quantification: Use fluorescently conjugated NAT10 antibodies to verify manipulation efficiency, then quantify global and transcript-specific ac4C levels using mass spectrometry or antibody-based methods.

• Stability assessments of target mRNAs: Combine actinomycin D chase experiments with RT-qPCR to determine how NAT10-mediated ac4C modification affects half-lives of target transcripts like MDR1 and BCRP .

• Polysome profiling: Assess how NAT10 expression and ac4C modification impact translation efficiency by analyzing mRNA distribution across polysome fractions.

• CRISPR-Cas9 mutational analysis: Generate catalytically inactive NAT10 mutants and use antibodies to confirm expression, then assess the impact on ac4C levels and target gene expression.

• Integration with clinical data: Correlate NAT10 expression detected by immunofluorescence with ac4C levels and expression of target genes in patient samples to validate findings from model systems.

• Live-cell imaging of mRNA dynamics: Combine MS2-tagged mRNA visualization with NAT10 antibody staining to track how NAT10-mediated acetylation affects mRNA localization and translation in real-time.

• Drug response correlation: Use NAT10 antibodies to monitor changes in protein expression during treatment with epigenetic modulators or RNA-targeting therapies, correlating with changes in ac4C profiles.

How might NAT10 antibodies contribute to the development of new cancer therapeutics?

NAT10 antibodies can support cancer therapeutic development through several approaches:

• Target validation: Use immunofluorescence and western blotting with NAT10 antibodies to confirm expression in cancer types being considered for NAT10-targeted therapies.

• Mechanism of action studies: Employ NAT10 antibodies to monitor protein levels, localization, and interactions during treatment with NAT10 inhibitors like remodelin, helping elucidate their mechanism of action.

• Pharmacodynamic biomarker development: Establish immunohistochemistry or flow cytometry protocols using NAT10 antibodies that could serve as pharmacodynamic biomarkers in clinical trials of NAT10 inhibitors.

• Combination therapy rational design: Use immunofluorescence to identify tumors with co-expression of NAT10 and potential synergistic targets, supporting the design of combination therapies.

• Patient selection strategies: Develop standardized immunohistochemistry or immunofluorescence protocols for identifying patients with high NAT10 expression who might benefit from targeted inhibition.

• Resistance monitoring: Track changes in NAT10 expression during treatment to identify potential resistance mechanisms and inform adaptive treatment strategies.

• Novel target identification: Combine NAT10 antibody-based immunoprecipitation with proteomic analysis to identify novel interacting partners that might themselves be therapeutic targets.

• Immuno-oncology applications: As NAT10 inhibition increases sensitivity to PD-1 therapy , NAT10 antibodies can help characterize the immunomodulatory effects of targeting this pathway and identify optimal combination approaches.

Recent research demonstrates the potential therapeutic value of targeting NAT10, as inhibition with remodelin overcame capecitabine resistance in breast cancer models both in vitro and in vivo , and increased sensitivity to PD-1 therapy in other cancer models .