NSUN2 Antibody, FITC conjugated

What is NSUN2 and why is it important in cancer research?

NSUN2 is an RNA methyltransferase that induces 5-methylcytosine (m5C) modification in mRNA, an important chemical posttranscriptional modification. It has been proven to play critical roles in the progression of various cancers, including osteosarcoma and anaplastic thyroid cancer. NSUN2 operates as a "writer" of m5C on target mRNAs, affecting their stability and expression levels. Higher expression of NSUN2 has been correlated with poorer prognosis in cancer patients, making it a potential prognostic marker and therapeutic target .

What are the optimal fixation and permeabilization methods for NSUN2-FITC antibody immunofluorescence?

For optimal results with NSUN2-FITC antibody in immunofluorescence:

• Fix cells with 4% paraformaldehyde for 15 minutes at room temperature

• Permeabilize with 0.2% Triton X-100 in PBS for 10 minutes

• Follow permeabilization with thorough washing (3× PBS, 5 minutes each)

• Block with 5% normal serum (matching secondary antibody host) for 1 hour

• Incubate with NSUN2-FITC antibody (1:100-1:500 dilution, optimized for your specific antibody) overnight at 4°C

This protocol preserves both cellular morphology and NSUN2 protein epitopes while allowing sufficient antibody penetration for accurate detection of nuclear NSUN2, which is critical when studying its methyltransferase activity in cancer cells .

How should I validate the specificity of NSUN2-FITC antibody in my experimental system?

A multi-step validation approach is recommended:

• Positive and negative controls: Use tissues/cells known to express (positive) or not express (negative) NSUN2

• Knockdown verification: Compare staining between NSUN2 knockdown/knockout cells and wild-type cells

• Western blot correlation: Confirm antibody detects a band of expected size (~86 kDa) in western blot

• Competitive binding assay: Pre-incubate antibody with recombinant NSUN2 protein before staining

• Cross-validation: Compare results with another validated NSUN2 antibody with a different epitope

In published research, NSUN2 knockdown in osteosarcoma cells showed significantly decreased expression levels, confirming antibody specificity for studying NSUN2-related pathways in cancer progression .

What controls should be included when studying NSUN2-mediated m5C modification?

When investigating NSUN2's m5C methyltransferase activity:

Studies have employed RNA immunoprecipitation (RIP) and methylated RIP to screen and validate NSUN2 targets, identifying FABP5 as a direct target in osteosarcoma cells. This methodological approach ensures reliable assessment of NSUN2's methyltransferase activity .

How can I optimize NSUN2-FITC antibody for dual immunofluorescence with other antibodies?

For successful dual immunofluorescence with NSUN2-FITC antibody:

• Spectral considerations: Pair FITC (excitation ~490nm, emission ~525nm) with fluorophores having minimal spectral overlap (e.g., Cy5, Texas Red)

• Sequential staining protocol:

  • First round: Apply NSUN2-FITC antibody (1:200 dilution) and incubate overnight at 4°C

  • Wash thoroughly (4× PBS, 5 minutes each)

  • Second round: Apply non-conjugated primary antibody followed by appropriate secondary antibody

• Signal balancing: Adjust acquisition settings to balance the typically strong nuclear NSUN2 signal with other targets

• Cross-reactivity prevention:

  • Block with 5% serum matching the host species of the second primary antibody

  • Include 0.1% Tween-20 in washing buffer to reduce non-specific binding

This approach has successfully demonstrated co-localization of NSUN2 with reader proteins like YBX1 in nuclei, providing valuable insights into m5C-dependent RNA processing mechanisms .

What are effective methodologies for studying the interaction between NSUN2 and its RNA targets using fluorescent antibodies?

To investigate NSUN2-RNA interactions:

• RNA Immunoprecipitation (RIP):

  • Cross-link protein-RNA complexes with 1% formaldehyde

  • Lyse cells and sonicate to fragment RNA

  • Immunoprecipitate with NSUN2-FITC antibody or separate NSUN2 antibody with magnetic beads

  • Reverse cross-links and isolate RNA

  • Perform RT-qPCR for suspected target RNAs (e.g., FABP5)

• Methylated RNA Immunoprecipitation (MeRIP):

  • Extract total RNA from cells

  • Fragment RNA and denature

  • Immunoprecipitate with m5C-specific antibody

  • Analyze enrichment by RNA sequencing or RT-qPCR

• Fluorescence in situ hybridization combined with immunofluorescence (FISH-IF):

  • Perform standard fixation and permeabilization

  • Hybridize with fluorescently labeled RNA probes for target RNA

  • Counter-stain with NSUN2-FITC antibody

  • Analyze co-localization using confocal microscopy

These techniques have successfully identified FABP5 as a direct target of NSUN2 in osteosarcoma cells, demonstrating that NSUN2 binds to and stabilizes FABP5 mRNA through m5C modification .

How can I analyze NSUN2 expression in relation to drug resistance phenotypes in clinical samples?

For correlating NSUN2 expression with drug resistance:

• Tissue microarray analysis:

  • Prepare tissue sections from drug-sensitive and resistant tumors

  • Perform immunofluorescence with NSUN2-FITC antibody

  • Quantify nuclear NSUN2 intensity using image analysis software

  • Correlate with patient treatment response data

• Single-cell RNA analysis with drug sensitivity correlation:

  • Process tumor samples for single-cell RNA sequencing

  • Cluster cells based on transcriptional profiles

  • Correlate NSUN2 expression with known drug resistance markers

  • Apply computational analysis to predict drug sensitivity

• Paired pre/post-treatment sample comparison:

  • Collect matched samples before and after treatment failure

  • Assess NSUN2 expression changes using immunofluorescence

  • Quantify changes in expression levels and cellular localization

Research has demonstrated that NSUN2 expression positively correlates with IC50 values of multiple anticancer agents, including both chemotherapy drugs and tyrosine kinase inhibitors, suggesting a role in multidrug resistance in cancer cells .

How should I address inconsistent staining results with NSUN2-FITC antibody?

When encountering variable NSUN2-FITC antibody staining:

For nuclear staining of NSUN2, ensure proper nuclear permeabilization, as NSUN2 is predominantly localized in the nucleus where it performs its methyltransferase activity on target RNAs .

What approaches help resolve contradictions between NSUN2 expression and observed phenotypes?

When experimental results contradict expected NSUN2-related phenotypes:

• Verify NSUN2 enzymatic activity:

  • Perform m5C dot blot assays to confirm methyltransferase activity

  • Use a catalytically inactive NSUN2 mutant as control

  • Check if total mRNA m5C levels decrease with NSUN2 knockdown

• Examine compensatory mechanisms:

  • Assess expression of other m5C methyltransferases (e.g., NSUN5, NSUN6)

  • Evaluate potential redundancy in RNA methylation pathways

• Validate downstream targets:

  • Confirm expression changes in known NSUN2 targets (e.g., FABP5)

  • Perform RIP-seq to identify cell type-specific RNA targets

• Context-dependent function analysis:

  • Investigate tissue/cell type-specific factors affecting NSUN2 function

  • Examine potential post-translational modifications altering NSUN2 activity

Research has shown that NSUN2 function can vary significantly between cancer types, with specific downstream targets like FABP5 in osteosarcoma and SRSF6 in anaplastic thyroid cancer, highlighting the importance of context-specific analysis .

How can NSUN2-FITC antibody be utilized to study m5C-dependent mRNA stability?

To investigate NSUN2's role in mRNA stability:

• RNA stability assay with NSUN2 modulation:

  • Treat cells with actinomycin D (5 μg/ml) to inhibit transcription

  • Harvest RNA at sequential timepoints (0, 1, 2, 4 hours)

  • Perform RT-qPCR to measure decay rate of target mRNAs

  • Compare stability between NSUN2 wildtype, knockdown, and overexpression conditions

• m5C reader protein analysis:

  • Use NSUN2-FITC for co-immunoprecipitation with potential reader proteins (e.g., YBX1)

  • Perform western blot to detect protein-protein interactions

  • Conduct RIP with YBX1 antibody to identify shared RNA targets

• Site-specific m5C analysis:

  • Employ bisulfite sequencing to map m5C sites in target mRNAs

  • Correlate methylation sites with NSUN2 binding regions

  • Create site-directed mutants to evaluate functional importance

Studies have demonstrated that NSUN2 stabilizes FABP5 mRNA through m5C modification, with YBX1 acting as a critical m5C reader that maintains mRNA stability. Knockdown of either NSUN2 or YBX1 decreased the stability and expression of FABP5 mRNA in osteosarcoma cells .

What experimental approaches can elucidate NSUN2's role in alternative splicing regulation?

To investigate NSUN2's impact on alternative splicing:

• Splicing reporter assays:

  • Design minigene constructs containing alternative exons

  • Transfect into cells with NSUN2 overexpression or knockdown

  • Analyze splicing patterns through RT-PCR

• RNA-seq for global splicing analysis:

  • Perform RNA-seq on NSUN2 modulated cells

  • Use computational tools (rMATS, VAST-TOOLS) to identify differential splicing events

  • Validate key events with RT-PCR

• CLIP-seq for splicing factor binding:

  • Conduct CLIP-seq for splicing factors (e.g., SRSF6) in NSUN2 wildtype vs. knockout cells

  • Map binding sites relative to alternatively spliced regions

  • Correlate with m5C modification sites

• Nuclear-cytoplasmic fractionation:

  • Separate nuclear and cytoplasmic fractions

  • Perform western blot with NSUN2-FITC antibody

  • Analyze distribution of splicing factors in relation to NSUN2 expression

Research has revealed that NSUN2 functions as a "writer" and ALYREF as a "reader" of m5C on SRSF6 mRNA, inducing alternative splicing reprogramming and redirecting the splice form of the UAP1 gene from AGX1 to AGX2 in anaplastic thyroid cancer .

How can NSUN2-FITC antibodies be used to investigate the relationship between m5C modification and cancer metabolism?

For studying NSUN2's role in cancer metabolism:

• Metabolic pathway analysis:

  • Use NSUN2-FITC antibody to isolate NSUN2-expressing cells via FACS

  • Perform metabolomics analysis on sorted populations

  • Compare metabolic profiles between NSUN2-high and NSUN2-low cells

• Fatty acid metabolism assessment:

  • Stain cells with NSUN2-FITC antibody and BODIPY 493/503 for neutral lipids

  • Quantify lipid content using flow cytometry or fluorescence microscopy

  • Compare lipid levels in NSUN2 wildtype vs. knockdown/knockout conditions

• Metabolic inhibitor studies:

  • Treat cells with metabolic inhibitors (e.g., Etomoxir for fatty acid oxidation)

  • Analyze NSUN2 expression and localization changes

  • Assess impact on m5C target mRNAs involved in metabolism

Research has demonstrated that NSUN2 promotes fatty acid metabolism in osteosarcoma cells by up-regulating FABP5 expression through m5C modification. NSUN2 knockdown led to accumulation of neutral lipids, while NSUN2 overexpression resulted in reduced neutral lipid content, highlighting a direct link between NSUN2 activity and lipid metabolism in cancer cells .

What approaches can be used to explore NSUN2 as a therapeutic target in multidrug-resistant cancers?

To investigate NSUN2 as a therapeutic target:

• NSUN2 inhibitor screening:

  • Develop high-throughput screening assays using NSUN2-FITC for binding displacement

  • Test small molecule compounds for inhibition of NSUN2 methyltransferase activity

  • Validate promising candidates with m5C dot blot assays

• Combination therapy assessment:

  • Treat drug-resistant cells with NSUN2 inhibitors alongside chemotherapy or targeted therapies

  • Monitor drug sensitivity using cell viability assays

  • Analyze expression of ABC transporters and drug efflux activity

• In vivo efficacy studies:

  • Establish xenograft models with NSUN2-high, drug-resistant tumors

  • Administer NSUN2 inhibitors alone or in combination with standard therapies

  • Assess tumor growth, m5C levels, and expression of NSUN2 target genes

• Biomarker development:

  • Use NSUN2-FITC antibody to establish NSUN2 expression thresholds that predict drug response

  • Correlate NSUN2 levels with resistance to specific therapeutic agents

  • Develop companion diagnostic approaches for patient stratification

Research has shown that NSUN2 expression correlates with multidrug resistance in anaplastic thyroid cancer, and NSUN2 inhibitors can reduce NSUN2 enzymatic activity and diminish downstream target expression, presenting a promising therapeutic approach to overcome MDR in cancer .

What are the key considerations for quantifying NSUN2 expression levels using FITC-conjugated antibodies?

For accurate quantification of NSUN2 using FITC-conjugated antibodies:

• Standardized acquisition parameters:

  • Establish fixed exposure settings for consistent signal detection

  • Include calibration standards in each experiment

  • Account for FITC photobleaching in time-course experiments

• Quantification methods:

  • Mean fluorescence intensity (MFI) for flow cytometry applications

  • Integrated density measurements for microscopy images

  • Nuclear:cytoplasmic ratio analysis for localization studies

• Normalization strategies:

  • Use housekeeping proteins as internal controls

  • Employ ratiometric analysis with stable reference fluorophores

  • Include biological reference samples across experimental batches

• Statistical approaches:

  • Apply appropriate statistical tests based on data distribution

  • Utilize correlation analyses for relating NSUN2 levels to phenotypic outcomes

  • Consider multivariate analyses when examining multiple parameters

Researchers have successfully used these approaches to demonstrate that higher NSUN2 expression predicts poorer prognosis in cancer patients and correlates with resistance to multiple anticancer agents, including both chemotherapy drugs and tyrosine kinase inhibitors .

How should researchers design experiments to investigate NSUN2's impact on drug resistance mechanisms?

For investigating NSUN2's role in drug resistance: