ythdf2 Antibody

What is YTHDF2 and why is it important to study?

YTHDF2 is a member of the YTH domain-containing family that specifically recognizes and binds to N6-methyladenosine (m6A)-modified RNAs. In humans, the canonical protein has 579 amino acid residues with a molecular mass of approximately 62.3 kDa . YTHDF2 is primarily involved in regulating mRNA stability through its binding to m6A-modified transcripts, promoting their degradation . This protein is important to study because:

• It plays critical roles in the cell cycle and innate immune responses

• It shows high expression in induced pluripotent stem cells (iPSCs) and is downregulated during neural differentiation

• It has been implicated in various cancers, including hepatocellular carcinoma

• It functions in neuronal development, including dendrite development in retinal ganglion cells

Understanding YTHDF2 function contributes to our knowledge of epitranscriptomic regulation and its implications in development and disease.

What applications are YTHDF2 antibodies commonly used for?

YTHDF2 antibodies are versatile tools employed in multiple immunodetection techniques:

The literature contains over 170 citations describing the use of YTHDF2 antibodies in research , demonstrating their established utility across multiple experimental approaches.

What is the subcellular localization of YTHDF2 and how can antibodies help determine this?

YTHDF2 exhibits dual localization in both the nucleus and cytoplasm . This distribution is critical to its function, as YTHDF2 helps relocalize bound mRNAs from the translatable pool to processing bodies (P-bodies), which are cytoplasmic mRNA decay sites .

To effectively determine YTHDF2 subcellular localization:

• Use immunofluorescence with dilutions of 1:50-1:800 depending on the antibody

• Include appropriate subcellular markers (e.g., DAPI for nuclei, markers for P-bodies)

• Consider cellular stress conditions, as YTHDF2 may relocalize under stress

• Use confocal microscopy for high-resolution localization studies

• Compare results across multiple cell types, as localization patterns may vary

Research has shown that under certain conditions, such as sodium arsenite treatment in HeLa cells, YTHDF2's localization pattern may change, which can be detected using immunofluorescence techniques .

How can researchers effectively use YTHDF2 antibodies to study its role in cancer progression?

Recent research has revealed complex and sometimes contradictory roles of YTHDF2 in different cancer types:

• Hepatocellular Carcinoma (HCC):

  • YTHDF2 acts as a tumor promoter by upregulating PD-L1 and VEGFA expression

  • It recognizes m6A modification in the 5'-untranslational region of ETV5 mRNA

  • This promotes HCC immune evasion and angiogenesis

• Lung Adenocarcinoma:

  • Contrary to its role in HCC, YTHDF2 inhibits migration and invasion of lung adenocarcinoma cells

  • It functions through regulating the FAM83D-TGFβ1-pSMAD2/3 pathway

  • YTHDF2 knockdown leads to decreased proliferation but increased invasion and migration capacity

For effective studies of YTHDF2 in cancer:

• Combine antibody-based approaches (IHC, IF, WB) with functional assays

• Perform RNA immunoprecipitation (RIP) to identify cancer-specific YTHDF2 mRNA targets

• Use tumor microarray analysis with YTHDF2 antibodies to correlate expression with clinical outcomes

• Consider dual staining with markers of immune cell infiltration to study immune evasion mechanisms

• Validate findings across multiple cancer cell lines and patient-derived samples

How should researchers approach YTHDF2 and m6A-related experimental design to resolve conflicting data in the literature?

The literature contains seemingly contradictory findings regarding YTHDF2 function:

To resolve such contradictions:

• Context-dependent analysis: Use YTHDF2 antibodies to assess expression across different cell types and tissues simultaneously

• Target validation: Perform RIP-seq followed by m6A-seq to identify cell-type-specific YTHDF2 targets

• Combinatorial knockdown: Compare phenotypes of single YTHDF2 knockdown with combined knockdown of multiple YTHDF family members

• Domain-specific studies: Use antibodies targeting different YTHDF2 domains to understand structure-function relationships

• Temporal dynamics: Examine YTHDF2 expression and localization across different time points and cellular states

Remember that YTHDF proteins may exhibit context-dependent functional compensation, as indicated in the literature , which could explain seemingly contradictory results.

What strategies should be employed when studying YTHDF2's role in immune responses?

YTHDF2 plays critical roles in modulating immune responses, particularly in tumor-associated macrophages (TAMs):

• YTHDF2 deficiency in TAMs suppresses tumor growth by reprogramming them toward an antitumoral phenotype

• It enhances their antigen cross-presentation ability, which increases CD8+ T cell-mediated antitumor immunity

• YTHDF2 in TAMs is regulated by interleukin-10–STAT3 signaling

• Targeting YTHDF2 in TAMs using TLR9 agonist-conjugated siRNA enhances cancer immunotherapy efficacy

For comprehensive immune-related studies:

• Macrophage polarization analysis:

  • Use YTHDF2 antibodies alongside markers of M1 (antitumoral) vs. M2 (protumoral) polarization

  • Monitor iNOS+/Arg1+ ratios in YTHDF2-sufficient vs. deficient macrophages

• T cell interaction studies:

  • Co-culture YTHDF2-deficient vs. control macrophages with T cells

  • Measure IFNγ production and T cell activation markers

• In vivo immune profiling:

  • Use flow cytometry with YTHDF2 antibodies to sort immune cell populations from tumor samples

  • Analyze YTHDF2 expression in correlation with immune checkpoint molecules

• Cytokine response analysis:

  • Examine how YTHDF2 affects IFNγ–STAT1 signaling in immune cells

  • Monitor changes in cytokine production in response to YTHDF2 modulation

What are the critical factors for successful Western blot analysis using YTHDF2 antibodies?

Western blot is the most commonly used application for YTHDF2 antibodies. For optimal results:

• Protein extraction and loading:

  • YTHDF2 has an expected molecular weight of 62.3 kDa but typically runs at 65-70 kDa on SDS-PAGE

  • Use appropriate lysis buffers that preserve protein integrity (e.g., RIPA buffer with protease inhibitors)

  • Load 20-50 μg of total protein per lane

• Antibody selection and dilution:

  • Primary antibody dilutions vary widely from 1:500 to 1:50,000 depending on the specific antibody

  • Polyclonal antibodies may detect additional bands compared to monoclonal antibodies

  • Consider antibodies validated for specific applications (e.g., knockdown/knockout validation)

• Controls and validation:

  • Include YTHDF2 knockdown/knockout samples as negative controls

  • Use cell lines with known YTHDF2 expression (e.g., HEK-293, Jurkat, HeLa, Raji cells)

  • Consider orthogonal validation methods such as RNAseq correlation

• Troubleshooting:

  • If observing multiple bands, verify isoform expression (up to 2 different isoforms reported)

  • For weak signals, extend exposure time or increase antibody concentration

  • For high background, increase blocking time or washing steps

How can researchers optimize immunoprecipitation protocols for studying YTHDF2-RNA interactions?

Studying YTHDF2-RNA interactions is crucial for understanding its functional role in m6A-mediated RNA regulation:

• RNA immunoprecipitation (RIP) protocol optimization:

  • Use 0.5-4.0 μg of YTHDF2 antibody per 1-3 mg of total protein lysate

  • Include RNase inhibitors in all buffers to preserve RNA integrity

  • Perform crosslinking (formaldehyde or UV) to capture transient interactions

  • Use low-salt wash conditions to preserve RNA-protein complexes

• Target validation approaches:

  • Perform RIP followed by RT-qPCR for candidate targets

  • For unbiased discovery, combine with RNA-seq (RIP-seq)

  • Validate findings with m6A-specific techniques (e.g., m6A-seq, miCLIP)

• Control experiments:

  • Include IgG control immunoprecipitations

  • Perform parallel IPs with other YTHDF family members to assess specificity

  • Include YTHDF2-depleted samples as negative controls

• Analysis of m6A-dependent binding:

  • Compare binding to transcripts with and without m6A modifications

  • Use METTL3/14 knockdown cells to reduce global m6A levels

  • Assess binding kinetics through time-course experiments

Studies have successfully used these approaches to identify YTHDF2 targets such as ETV5 in hepatocellular carcinoma and Kalrn, Strn, and Ubr4 in retinal ganglion cells .

What considerations are important when selecting YTHDF2 antibodies for different experimental applications?

Different experimental applications require careful antibody selection:

When selecting antibodies:

• Epitope considerations:

  • N-terminal antibodies target the proline/glutamine-rich region involved in localization

  • C-terminal antibodies target the YTH domain responsible for m6A binding

  • Consider using antibodies targeting different regions for confirmatory experiments

• Validation methods:

  • Orthogonal RNAseq validation compares antibody performance with transcript expression

  • Review validation data galleries provided by suppliers

  • Consider enhanced validation methodologies like immunogen-specific blocking

• Species reactivity:

  • YTHDF2 is conserved across species, with orthologs reported in mouse, rat, bovine, frog, chimpanzee, and chicken

  • Many antibodies show cross-reactivity across species (e.g., human, mouse, rat)

  • Verify sequence homology in the epitope region when working with non-standard model organisms

• Application-specific recommendations:

  • For challenging applications like IHC, consider antibodies specifically validated for this purpose

  • For novel applications, test multiple antibodies targeting different epitopes

  • For quantitative applications, use recombinant antibodies for batch-to-batch consistency

How can YTHDF2 antibodies be used to explore therapeutic targeting of this protein in cancer?

Recent research suggests YTHDF2 as a potential therapeutic target in certain cancers:

• Small molecule inhibitor development:

  • Phenylpyrazoles like CK-75 have been identified as YTHDF2 inhibitors that disrupt m6A-YTHDF2 binding

  • Antibodies can serve as critical tools for validating target engagement in inhibitor screening

  • Use YTHDF2 antibodies in cellular thermal shift assays (CETSA) to confirm compound binding

• RNA-based therapeutic approaches:

  • CpG oligodeoxynucleotide-conjugated YTHDF2 siRNAs target macrophages and enhance PD-L1 antibody therapy

  • YTHDF2 antibodies can validate knockdown efficiency and cellular effects

  • Monitor pathway modulation (e.g., IFNγ-STAT1 signaling) following therapeutic targeting

• Biomarker development:

  • YTHDF2 antibodies can assess expression levels in patient samples

  • Correlate expression with treatment response and patient outcomes

  • Develop companion diagnostics for YTHDF2-targeting therapies

• Combination therapy strategies:

  • YTHDF2 targeting combined with immune checkpoint inhibitors shows promise

  • Use antibody-based techniques to monitor both YTHDF2 and immune checkpoint expression

  • Analyze tumor microenvironment changes following combination treatment

In hepatocellular carcinoma, targeting YTHDF2 via siRNA-containing aptamer/liposomes successfully inhibited immune evasion, suggesting promising therapeutic applications .

What are the best approaches for studying YTHDF2 in neuronal development using antibodies?

YTHDF2 plays important roles in neuronal development, particularly in dendrite formation:

• Retinal ganglion cell (RGC) development:

  • YTHDF2 acts as a negative regulator for dendrite development in RGCs

  • Knockdown or knockout of YTHDF2 leads to increased stability of target mRNAs

  • Key targets include Kalrn, Strn, and Ubr4, which show increased protein levels in YTHDF2-deficient retinas

For studying YTHDF2 in neuronal contexts:

• Immunohistochemistry optimization:

  • Use antigen retrieval methods (e.g., TE buffer pH 9.0 or citrate buffer pH 6.0)

  • Combine with neuronal markers for co-localization studies

  • Consider tissue-specific fixation protocols for optimal epitope preservation

• Developmental time-course analysis:

  • Use YTHDF2 antibodies to track expression changes during neuronal differentiation

  • Correlate with markers of neuronal maturation

  • Compare with expression of other m6A regulatory proteins

• Target validation in neuronal contexts:

  • Perform RIP-seq in neuronal tissues or differentiated cells

  • Validate target regulation using reporter assays

  • Examine stability of identified target mRNAs following YTHDF2 manipulation

• Functional analysis:

  • Use morphological assessments combined with YTHDF2 immunostaining

  • Quantify dendrite complexity in YTHDF2 wildtype vs. deficient neurons

  • Examine YTHDF2 localization in dendrites and synapses

Research has demonstrated that YTHDF2 controls the protein levels of its m6A-modified target mRNAs by decreasing their stability, which directly impacts neuronal morphology and function .