YTHDF2 Antibody

What is YTHDF2 and why is it important in cellular biology?

YTHDF2 (YTH N6-methyladenosine RNA binding protein F2) is a critical m6A reader protein comprising 579 amino acid residues with a mass of 62.3 kDa in humans. It is localized in both the nucleus and cytoplasm, with up to two different isoforms reported. YTHDF2 recognizes m6A-modified RNA and typically promotes the degradation of its target transcripts, playing a vital role in RNA metabolism. It is highly expressed in induced pluripotent stem cells (iPSCs) and becomes downregulated during neural differentiation. As a member of the YTHDF protein family, YTHDF2 is involved in cell cycle regulation and innate immune responses. Several synonyms exist for this protein, including DF2, HGRG8, NY-REN-2, and YTH domain-containing family protein 2 .

What types of YTHDF2 antibodies are available for research applications?

Several types of YTHDF2 antibodies are available for research use, including polyclonal and monoclonal formats. Polyclonal antibodies from various suppliers (Biomatik, EpiGentek, OriGene Technologies) are commonly available, recognizing different epitopes of the YTHDF2 protein. Additionally, recombinant rabbit monoclonal antibodies are available from providers like Starter Biotechnology. Most commercial antibodies are unconjugated, though some suppliers may offer custom conjugation. These antibodies have been validated for various applications including Western Blot (WB), ELISA, Immunofluorescence (IF), and Immunohistochemistry (IHC), with a range of reactivity across species including human, mouse, rabbit, rat, and others .

What are the key applications of YTHDF2 antibodies in research?

YTHDF2 antibodies serve multiple critical research applications:

• Western Blot: For detecting and quantifying YTHDF2 protein expression

• Immunohistochemistry: For visualizing YTHDF2 distribution in tissue sections

• Immunofluorescence: For subcellular localization studies

• ELISA: For quantitative measurement in solution

• RNA Immunoprecipitation (RIP): For identifying YTHDF2-bound RNAs

• Chromatin Immunoprecipitation (ChIP): For studying YTHDF2 interactions with chromatin

Over 170 citations in scientific literature document the use of YTHDF2 antibodies, with Western Blot being the most widely reported application .

How should YTHDF2 antibodies be stored and handled for optimal performance?

For optimal performance and longevity, YTHDF2 antibodies should be stored according to manufacturer recommendations, typically at -20°C for long-term storage and 4°C for short-term use. Avoid repeated freeze-thaw cycles by aliquoting the antibody before freezing. When handling, maintain sterile conditions and use appropriate buffers (typically PBS with 0.1% sodium azide). For dilution, use manufacturer-recommended buffers, often containing 1-5% BSA or non-fat milk. During experiments, keep antibodies on ice when in use and return to refrigeration promptly. Always include proper controls when using these antibodies, such as positive control samples with known YTHDF2 expression and negative controls where the antibody is omitted or blocked with a specific peptide .

What is the optimal protocol for using YTHDF2 antibodies in Western Blot analysis?

For optimal Western Blot analysis using YTHDF2 antibodies, follow this detailed protocol:

• Sample preparation: Extract protein from cells/tissues using RIPA buffer containing protease inhibitors. Quantify protein concentration using BCA or Bradford assay.

• SDS-PAGE: Separate 20-40 μg of protein on an 8-12% gel, including a molecular weight marker.

• Transfer: Transfer proteins to a PVDF membrane at 100V for 60-90 minutes in cold transfer buffer.

• Blocking: Block membrane with 5% non-fat milk or BSA in TBST for 1 hour at room temperature.

• Primary antibody incubation: Dilute YTHDF2 antibody (typically 1:1000-1:5000, check specific product recommendations) in blocking solution and incubate overnight at 4°C with gentle rocking.

• Washing: Wash membrane 3-4 times with TBST, 5-10 minutes each.

• Secondary antibody: Incubate with HRP-conjugated secondary antibody (1:5000-1:10000) for 1 hour at room temperature.

• Washing: Repeat washing steps.

• Detection: Apply ECL substrate and image using a digital imager.

• Analysis: Normalize YTHDF2 signal to a loading control (β-actin or GAPDH) for quantification.

When analyzing results, YTHDF2 should appear as a band at approximately 62.3 kDa. Variation in band size may indicate detection of different isoforms or post-translational modifications .

How can researchers effectively design RNA immunoprecipitation (RIP) assays using YTHDF2 antibodies?

For effective YTHDF2 RNA immunoprecipitation (RIP) assays, follow this methodological approach:

• Cell preparation:

  • Collect cells from 2-3 15-cm plates (approximately 1-2×10^7 cells)

  • Wash with cold PBS and pellet by centrifugation at 1,000g for 5 minutes

• Cell lysis:

  • Resuspend cells in lysis buffer 1 (0.5% SDS in PBS with protease inhibitors and RNase inhibitors at 400 U/ml)

  • Incubate on ice for 20 minutes

  • Add lysis buffer 2 (0.2% Triton-100 in PBS with protease and RNase inhibitors)

  • Incubate on ice for another 20 minutes

  • Centrifuge at 14,000g for 20 minutes to remove debris

• Immunoprecipitation:

  • Pre-clear lysate with Protein A/G beads for 1 hour at 4°C

  • Incubate cleared lysate with anti-YTHDF2 antibody (5-10 μg) at 4°C overnight with rotation

  • Add pre-washed Protein A/G magnetic beads (100 μl) and incubate for 4 hours at 4°C

  • Use IgG as negative control

• Washing and RNA extraction:

  • Wash beads 4-5 times with wash buffer

  • Extract RNA using TRIzol reagent followed by chloroform extraction

  • Precipitate RNA with isopropanol and wash with ethanol

  • Resuspend in RNase-free water

• Analysis:

  • Perform RT-qPCR for target mRNAs

  • Include non-target mRNAs (like actin) as negative controls

  • Calculate enrichment relative to input and IgG control

This protocol has been validated for identifying direct YTHDF2 mRNA targets such as FAM83D in lung adenocarcinoma research . Important considerations include maintaining RNase-free conditions throughout and using appropriate controls to confirm specificity of the interaction.

What are the optimal conditions for immunofluorescence staining using YTHDF2 antibodies?

For optimal immunofluorescence staining with YTHDF2 antibodies, implement the following protocol:

• Cell preparation:

  • Culture cells on sterile coverslips in appropriate medium

  • When cells reach 60-70% confluence, proceed to fixation

• Fixation and permeabilization:

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

  • Wash 3 times with PBS (5 minutes each)

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

  • Wash 3 times with PBS

• Blocking:

  • Block with 5% normal serum (from the same species as secondary antibody) in PBS for 1 hour at room temperature

• Primary antibody incubation:

  • Dilute YTHDF2 antibody (typically 1:100-1:500, verify with manufacturer) in blocking solution

  • Incubate overnight at 4°C in a humidified chamber

  • For dual staining, include other primary antibodies against subcellular markers

• Washing:

  • Wash 4 times with PBS (5 minutes each)

• Secondary antibody incubation:

  • Incubate with fluorophore-conjugated secondary antibody (1:500-1:1000) for 1 hour at room temperature in the dark

  • For nuclear visualization, include DAPI (1:1000) during the last 10 minutes

• Final washing and mounting:

  • Wash 4 times with PBS (5 minutes each)

  • Mount coverslips on slides using anti-fade mounting medium

• Imaging:

  • Image using a confocal or fluorescence microscope

When interpreting results, expect to observe YTHDF2 in both nuclear and cytoplasmic compartments, with potential variation depending on cell type and experimental conditions. For quantitative analysis, use appropriate software to measure fluorescence intensity and co-localization with other proteins of interest .

What controls should be included when using YTHDF2 antibodies for experimental validation?

When using YTHDF2 antibodies for experimental validation, incorporate these essential controls:

• Positive controls:

  • Cell lines or tissues with confirmed YTHDF2 expression (e.g., A549 or H1299 lung adenocarcinoma cells)

  • Recombinant YTHDF2 protein for antibody validation

  • Overexpression system with YTHDF2 plasmid

• Negative controls:

  • Isotype control antibody (same species and concentration as YTHDF2 antibody)

  • Secondary antibody-only control (omit primary antibody)

  • YTHDF2 knockdown samples (siRNA or shRNA-treated cells)

  • Peptide competition assay (pre-incubate antibody with immunizing peptide)

• Loading/processing controls:

  • Housekeeping protein detection (β-actin, GAPDH) for Western blot

  • Total protein stain (Ponceau S) for membrane loading verification

  • Non-target mRNA (actin) for RIP experiments

• Technical controls:

  • Biological replicates (minimum three independent experiments)

  • Multiple antibody concentrations for optimization

  • Different YTHDF2 antibodies targeting distinct epitopes

• Specificity controls:

  • Cross-reactivity assessment with related proteins (YTHDF1, YTHDF3)

  • Testing in multiple cell lines/tissues

Implementation of these controls ensures reliable and reproducible results while minimizing false positives and negatives. For functional studies, complementary approaches like correlation between protein level (by antibody detection) and mRNA level (by qPCR) provide additional validation .

How does YTHDF2 expression vary across different cancer types?

YTHDF2 expression shows significant variation across cancer types, with distinct patterns of dysregulation:

In lung adenocarcinoma specifically, proteomic analysis of 103 patient samples in the CPTAC cohort confirmed elevated YTHDF2 expression in tumor tissues compared to adjacent normal tissues. Subgroup analyses revealed that this elevated expression pattern was consistent across most patient demographics, including different sexes, most age groups, and TNM stages I-III, though the difference was not significant in patients younger than 40 years or with stage IV disease .

The varying expression patterns suggest that YTHDF2 may play cancer-type specific roles, potentially functioning as either an oncogene or tumor suppressor depending on the cellular context and cancer type. These findings highlight the importance of cancer-specific investigations when studying YTHDF2's role in oncogenesis and progression .

What methodologies can be used to study YTHDF2's impact on cancer cell migration and invasion?

To study YTHDF2's impact on cancer cell migration and invasion, researchers can employ these methodological approaches:

• YTHDF2 expression modulation:

  • Knockdown: Design and transfect specific siRNAs targeting YTHDF2 (verify knockdown efficiency via Western blot and qRT-PCR)

  • Overexpression: Transfect cells with YTHDF2-expressing plasmids

  • CRISPR/Cas9: Generate stable YTHDF2 knockout cell lines

• Migration assays:

  • Wound healing/scratch assay: Create a cell-free zone in a confluent monolayer, image at regular intervals (0, 24, 48h), and calculate closure rate

  • Boyden chamber assay: Place cells in serum-free medium in upper chamber, with full medium in lower chamber; quantify cells migrating through membrane

• Invasion assays:

  • Transwell Matrigel invasion assay: Coat Transwell chambers with Matrigel matrix, culture cells in FBS-free medium in upper chamber with DMEM+15% FBS in lower chamber; after 48h, fix, stain with crystal violet, and quantify invaded cells

  • 3D spheroid invasion assay: Form cancer cell spheroids, embed in Matrigel, and monitor invasion into surrounding matrix

• Molecular mechanism investigation:

  • EMT marker analysis: Assess expression changes in E-cadherin, N-cadherin, vimentin, and Snail via Western blot

  • RIP assay: Identify YTHDF2-bound mRNAs related to migration/invasion

  • m6A-seq: Map m6A modifications in migration/invasion-related transcripts

• In vivo validation:

  • Metastasis models: Inject YTHDF2-modulated cells into mice and evaluate metastatic potential

In lung adenocarcinoma research, these approaches revealed that YTHDF2 knockdown led to increased migration and invasion capabilities in A549 and H1299 cell lines, while simultaneously decreasing proliferation. This was demonstrated through wound healing assays showing faster wound closure and Transwell invasion assays showing increased invasive capacity in YTHDF2-silenced cells. Western blot analysis of EMT markers further supported these findings, suggesting YTHDF2 functions as a migration and invasion suppressor in lung adenocarcinoma .

How can researchers investigate the relationship between YTHDF2 and immune evasion in cancer?

To investigate YTHDF2's role in cancer immune evasion, researchers should implement this comprehensive methodological framework:

• Expression correlation analysis:

  • Analyze transcriptomic and proteomic datasets to correlate YTHDF2 expression with immune checkpoint molecules (PD-L1, CTLA-4, etc.)

  • Perform immunohistochemistry on patient samples to evaluate co-expression patterns

  • Use flow cytometry to quantify simultaneous expression of YTHDF2 and immune checkpoint proteins

• Functional modulation studies:

  • Generate YTHDF2 knockdown and overexpression cancer cell models

  • Assess changes in immune checkpoint molecule expression (PD-L1, PD-L2, etc.) via Western blot, qRT-PCR, and flow cytometry

  • Perform RNA stability assays to determine if YTHDF2 affects mRNA stability of immune regulators

• Mechanistic investigation:

  • Conduct RIP assays to identify direct binding between YTHDF2 and mRNAs of immune-related genes

  • Perform m6A-seq to map m6A modifications in immune checkpoint transcripts

  • Investigate downstream signaling pathways using phosphorylation-specific antibodies

• Co-culture experiments:

  • Establish co-culture systems with YTHDF2-modified cancer cells and immune cells (T cells, NK cells)

  • Measure immune cell activation, cytokine production, and cytotoxicity against cancer cells

  • Assess T cell proliferation and activation marker expression (CD69, CD25) by flow cytometry

• In vivo models:

  • Generate syngeneic mouse models with YTHDF2-modified cancer cells

  • Analyze tumor-infiltrating lymphocytes by flow cytometry and immunohistochemistry

  • Test combination therapies with immune checkpoint inhibitors

In hepatocellular carcinoma research, this approach revealed that YTHDF2 acts as a tumor promoter by upregulating PD-L1 and VEGFA expression. Mechanistically, YTHDF2 was found to recognize m6A-modified ETV5 mRNA, recruiting eIF3b to facilitate its translation. Elevated ETV5 then induced transcription of PD-L1 and VEGFA, promoting immune evasion and angiogenesis. These findings suggest YTHDF2 as a potential therapeutic target for HCC treatment .

What techniques can be used to identify and validate YTHDF2 mRNA targets in cancer cells?

To identify and validate YTHDF2 mRNA targets in cancer cells, implement this multi-technique approach:

• Transcriptome-wide identification:

  • RIP-seq: Immunoprecipitate YTHDF2-bound RNAs using validated antibodies, followed by RNA sequencing

  • CLIP-seq or PAR-CLIP: UV crosslink YTHDF2 to bound RNAs before immunoprecipitation for higher specificity

  • m6A-seq: Map m6A modifications across the transcriptome to identify potential YTHDF2 binding sites

  • RNA-seq after YTHDF2 knockdown/overexpression: Identify differentially expressed transcripts

• Computational analysis:

  • Motif enrichment analysis to identify common sequence features

  • GO and pathway enrichment to identify functional categories

  • Integration with m6A databases to filter for methylated transcripts

• Direct binding validation:

  • Targeted RIP-qPCR: Perform RIP using YTHDF2 antibodies followed by qPCR for candidate targets

  • RNA pull-down assays: Use biotinylated RNA probes of candidate targets to capture YTHDF2 protein

  • Luciferase reporter assays with wild-type and mutated m6A sites

• Functional validation:

  • RNA stability assays: Measure half-life of target mRNAs after transcription inhibition (actinomycin D) in YTHDF2 knockdown/overexpression cells

  • Polysome profiling: Assess translation efficiency of target mRNAs

  • Target protein expression analysis via Western blot

• In vivo confirmation:

  • Rescue experiments: Restore phenotype by modulating target gene expression in YTHDF2-altered cells

This approach was successfully applied to identify FAM83D as a direct YTHDF2 target in lung adenocarcinoma. RIP assays confirmed YTHDF2 binding to FAM83D mRNA, while knockdown of YTHDF2 resulted in significantly increased FAM83D mRNA and protein levels. This established that YTHDF2 inhibits FAM83D expression by promoting the degradation of its m6A-modified mRNA, demonstrating the regulatory effect of the YTHDF2-FAM83D pathway in cancer progression .

How can YTHDF2 antibodies be used in combination with other techniques to study m6A modifications?

YTHDF2 antibodies can be integrated with multiple complementary techniques to comprehensively study m6A modifications:

• Integrated m6A-seq and YTHDF2 RIP-seq:

  • Perform m6A-seq to map global m6A modifications

  • Conduct YTHDF2 RIP-seq to identify YTHDF2-bound transcripts

  • Integrate datasets to identify m6A-modified transcripts specifically recognized by YTHDF2

  • Analyze overlapping transcripts using pathway enrichment tools

• Proximity ligation assays (PLA):

  • Use anti-YTHDF2 antibody alongside antibodies against m6A writers (METTL3/14) or erasers (FTO, ALKBH5)

  • Visualize and quantify protein-protein interactions in situ

  • Assess how these interactions change under different cellular conditions

• YTHDF2-APEX2 proximity labeling:

  • Generate YTHDF2-APEX2 fusion proteins

  • Use biotin-phenol labeling to identify proteins in close proximity to YTHDF2

  • Analyze the interactome to identify novel m6A regulation machinery components

• CRISPR-dCas13 with YTHDF2 antibodies:

  • Target specific m6A sites with dCas13 fused to m6A writers/erasers

  • Use YTHDF2 immunoprecipitation to confirm altered binding after m6A modification

• Single-molecule RNA tracking with YTHDF2 detection:

  • Label target mRNAs with MS2 or similar systems

  • Use fluorescently tagged YTHDF2 antibodies in fixed cells or YTHDF2-GFP in live cells

  • Track co-localization to monitor dynamic YTHDF2-mRNA interactions

• RNA structure probing with YTHDF2 footprinting:

  • Perform SHAPE-MaP or similar structure probing with and without YTHDF2 binding

  • Map structural changes induced by YTHDF2 recognition of m6A sites

These integrated approaches provide comprehensive insights into how YTHDF2 specifically recognizes m6A-modified transcripts, how these interactions impact RNA fate, and the regulatory networks controlling these processes. This multi-technique strategy has been instrumental in identifying critical YTHDF2 targets like FAM83D in cancer research and ETV5 in hepatocellular carcinoma .

What approaches can researchers use to study YTHDF2's role in post-transcriptional regulation?

To investigate YTHDF2's role in post-transcriptional regulation, researchers should implement these methodological approaches:

• RNA stability and decay analysis:

  • Actinomycin D chase assays: Treat YTHDF2 knockdown/overexpression cells with actinomycin D to block transcription, then measure target mRNA levels at multiple timepoints by qRT-PCR

  • Bromouridine (BrU) pulse-chase: Label newly synthesized RNA with BrU, immunoprecipitate at different timepoints, and quantify specific transcripts

  • SLAM-seq (thiol(SH)-linked alkylation for the metabolic sequencing of RNA): Measure nucleotide conversion rates to determine RNA decay rates genome-wide

• Translation regulation assessment:

  • Polysome profiling: Fractionate ribosomes on sucrose gradients and analyze distribution of target mRNAs across non-translating, monosome, and polysome fractions

  • Ribosome profiling: Sequence ribosome-protected fragments to measure translation efficiency

  • Puromycin incorporation assays: Measure global protein synthesis rates

  • Luciferase reporter assays with 5'UTR, coding region, or 3'UTR of target genes

• Protein-RNA interaction dynamics:

  • FRAP (Fluorescence Recovery After Photobleaching): Measure kinetics of YTHDF2 binding to RNA

  • BiFC (Bimolecular Fluorescence Complementation): Visualize interactions between YTHDF2 and RNA decay machinery

  • IP-MS (Immunoprecipitation-Mass Spectrometry): Identify protein complexes associated with YTHDF2

• Subcellular localization studies:

  • Immunofluorescence with YTHDF2 antibodies and markers for processing bodies (P-bodies), stress granules, or other RNA granules

  • Co-localization analysis with RNA decay machinery components (DCP1/2, XRN1, exosome)

  • Live-cell imaging of fluorescently tagged YTHDF2 and target mRNAs

• Global effect assessment:

  • YTHDF2 eCLIP-seq: Map transcriptome-wide binding sites with single-nucleotide resolution

  • Paired RNA-seq and proteomics: Correlate changes in mRNA and protein levels after YTHDF2 modulation

This multifaceted approach has revealed that YTHDF2 can selectively regulate the stability of specific mRNAs like FAM83D in lung adenocarcinoma cells. When YTHDF2 was knocked down, researchers observed significant increases in FAM83D mRNA and protein levels, confirming YTHDF2's role in promoting the degradation of its target transcripts through recognition of m6A modifications .

How can researchers design experiments to differentiate between YTHDF1, YTHDF2, and YTHDF3 functions?

To differentiate between YTHDF1, YTHDF2, and YTHDF3 functions, researchers should implement the following experimental design strategy:

• Selective manipulation approaches:

  • Individual knockdown: Design specific siRNAs/shRNAs targeting unique regions of each YTHDF protein

  • Combinatorial knockdown: Create double and triple knockdowns to identify redundant/synergistic effects

  • Rescue experiments: Express one YTHDF protein in triple-knockdown background

  • Domain swapping: Create chimeric proteins with YTH domains exchanged between family members

• Distinctive functional assays:

  • RNA stability (YTHDF2-focused): Actinomycin D chase experiments monitoring mRNA half-life

  • Translation efficiency (YTHDF1-focused): Polysome profiling, ribosome profiling, and puromycin incorporation assays

  • Subcellular localization (YTHDF3-focused): RNA granule formation and P-body localization studies

• Binding specificity analysis:

  • Parallel RIP-seq or CLIP-seq for all three proteins under identical conditions

  • Motif analysis to identify unique binding preferences

  • Competition binding assays with recombinant proteins

• Temporal dynamics investigation:

  • Time-course experiments after cellular stimulation

  • Pulse-chase labeling to track RNA fate

  • Live-cell imaging with differentially tagged YTHDF proteins

• Protein interaction network mapping:

  • BioID or APEX proximity labeling for each YTHDF protein

  • Co-immunoprecipitation with mass spectrometry to identify unique interaction partners

  • Yeast two-hybrid screening

• Substrate specificity determination:

  • RNA immunoprecipitation coupled to high-throughput sequencing (RIP-seq)

  • Cross-linking immunoprecipitation (CLIP-seq)

  • RNA Bind-n-Seq to determine binding motifs

This comprehensive approach has revealed distinct functions: YTHDF1 primarily enhances translation efficiency, YTHDF2 predominantly promotes mRNA decay, and YTHDF3 cooperates with both YTHDF1 and YTHDF2 to modulate their functions. In cancer research, this differentiation is crucial, as YTHDF2 has been shown to inhibit migration and invasion in lung adenocarcinoma while promoting tumor growth in hepatocellular carcinoma, highlighting context-dependent roles that may differ from its YTHDF family counterparts .

What are common problems when using YTHDF2 antibodies and how can they be addressed?

Common problems with YTHDF2 antibodies and their solutions include:

• High background in Western blots:

  • Problem: Non-specific binding resulting in multiple bands or smeared signals

  • Solutions:

    • Increase blocking time/concentration (5% milk or BSA for 2 hours)

    • Use more stringent washing (0.1-0.3% Tween-20 in TBS)

    • Titrate antibody concentration (try 1:2000-1:5000 dilutions)

    • Add 0.1% SDS to antibody diluent to reduce non-specific binding

    • Use fresher antibody aliquots to avoid aggregation

• Weak or no signal:

  • Problem: Insufficient antibody binding or low target protein expression

  • Solutions:

    • Increase protein loading (50-80 μg per lane)

    • Optimize antibody concentration (try 1:500-1:1000)

    • Extend primary antibody incubation (overnight at 4°C)

    • Use enhanced detection systems (high-sensitivity ECL)

    • Include positive control (A549 or H1299 cells known to express YTHDF2)

    • Verify protein transfer efficiency with Ponceau S staining

• Cross-reactivity with other YTHDF family members:

  • Problem: Antibody detecting YTHDF1 or YTHDF3 due to sequence homology

  • Solutions:

    • Use antibodies targeting unique regions (C-terminal domain rather than YTH domain)

    • Validate specificity using YTHDF2 knockdown samples

    • Perform peptide competition assays

    • Use recombinant YTHDF proteins as controls

• Inconsistent RIP results:

  • Problem: Variable RNA enrichment between experiments

  • Solutions:

    • Maintain strict RNase-free conditions

    • Optimize crosslinking parameters

    • Include RNase inhibitors (400 U/ml) throughout the protocol

    • Verify antibody immunoprecipitation efficiency before proceeding to RNA extraction

    • Standardize lysate input and antibody amounts

• Immunofluorescence non-specific staining:

  • Problem: Diffuse or unexpected staining patterns

  • Solutions:

    • Increase blocking time (2 hours at room temperature)

    • Add 0.1-0.3% Triton X-100 to antibody diluent

    • Use Sudan Black B (0.1% in 70% ethanol) to reduce autofluorescence

    • Include YTHDF2 siRNA-treated cells as negative controls

    • Optimize fixation method (try 2-4% PFA or methanol)

Implementing these troubleshooting approaches has been effective in research studying YTHDF2's role in lung adenocarcinoma and hepatocellular carcinoma, where specific and reliable antibody performance was crucial for identifying authentic YTHDF2-RNA interactions and protein expression patterns .

How should researchers interpret contradictory results about YTHDF2's role in different cancer types?

When interpreting contradictory results about YTHDF2's role across different cancer types, researchers should implement this systematic evaluation framework:

For example, in lung adenocarcinoma, YTHDF2 inhibits migration and invasion while decreasing proliferation, suggesting a context-specific tumor suppressor role mediated through FAM83D regulation . In contrast, in hepatocellular carcinoma, YTHDF2 acts as a tumor promoter by upregulating PD-L1 and VEGFA expression through ETV5 mRNA regulation, facilitating immune evasion and angiogenesis . These contradictory roles likely reflect tissue-specific target mRNA populations and signaling contexts rather than inconsistent experimental approaches.

What considerations are important when analyzing YTHDF2 expression data from patient samples?

When analyzing YTHDF2 expression data from patient samples, researchers should consider these critical factors:

• Sample quality and preparation considerations:

  • Tissue preservation method (FFPE vs. fresh-frozen) affects RNA/protein quality

  • Ischemic time before fixation influences protein degradation

  • Tumor heterogeneity requires multiple sampling regions

  • Normal adjacent tissue may harbor molecular alterations despite histological normalcy

  • Patient treatment history may affect YTHDF2 expression patterns

• Technical and analytical factors:

  • Antibody selection: Different antibodies may target different epitopes or isoforms

  • Detection method sensitivity: IHC vs. Western blot vs. proteomic approaches

  • Scoring systems: H-score, percentage positive cells, or intensity scales affect interpretation

  • Batch effects in multi-center studies require normalization

  • Subcellular localization (nuclear vs. cytoplasmic) provides functional insights

• Patient stratification considerations:

  • Demographic factors: Age, sex, ethnicity influence baseline expression

  • Tumor stage and grade correlate with expression patterns

  • Molecular subtypes may show distinct YTHDF2 dependencies

  • Treatment history affects expression profiles

  • Comorbidities influence tumor microenvironment and expression

• Statistical analysis approaches:

  • Appropriate cutoff determination: ROC curve analysis for optimal thresholds

  • Multiple testing correction for genome-wide studies

  • Survival analysis methods: Kaplan-Meier with log-rank tests and Cox regression

  • Multivariate models to account for confounding variables

  • Power calculations to ensure adequate sample size

• Validation strategies:

  • Independent cohort validation

  • Multi-platform confirmation (mRNA and protein)

  • Single-cell analyses to address heterogeneity

  • Functional validation in patient-derived models

What emerging technologies might enhance YTHDF2 antibody-based research?

Emerging technologies poised to enhance YTHDF2 antibody-based research include:

• Advanced proximity labeling methods:

  • TurboID and miniTurbo: Faster biotin ligases for more temporally precise interactome mapping

  • Split-TurboID: For detecting conditional YTHDF2 interactions based on specific cellular events

  • APEX2-mediated proximity labeling: For capturing transient YTHDF2-protein interactions in living cells

  • Application: Identify novel YTHDF2 interaction partners in specific cellular compartments or under various stresses

• Super-resolution microscopy techniques:

  • STORM/PALM: For visualizing YTHDF2 localization with ~20nm resolution

  • Expansion microscopy: Physical expansion of specimens for improved optical resolution

  • Lattice light-sheet microscopy: For rapid 3D imaging of YTHDF2 dynamics in living cells

  • Application: Track real-time movement of YTHDF2-mRNA complexes to processing bodies or stress granules

• Single-molecule approaches:

  • Single-molecule FRET: For studying YTHDF2-RNA binding dynamics

  • smFISH combined with immunofluorescence: For correlating YTHDF2 localization with specific target mRNAs

  • Optical tweezers with antibody-coated beads: For measuring YTHDF2-RNA binding forces

  • Application: Determine kinetic parameters of YTHDF2 binding to various RNA substrates

• Nanobody and aptamer technologies:

  • YTHDF2-specific nanobodies: Smaller probes for improved tissue penetration and reduced immunogenicity

  • RNA aptamers targeting YTHDF2: For intracellular tracking without antibodies

  • Application: Live-cell imaging of endogenous YTHDF2 without transfection

• Spatial transcriptomics with antibody detection:

  • MERFISH combined with immunofluorescence: For correlating YTHDF2 protein localization with its target mRNAs

  • Visium spatial transcriptomics with protein detection: For mapping YTHDF2 activity across tissue regions

  • Application: Create spatial maps of YTHDF2-regulated transcripts in tumor microenvironments

• Microfluidic and single-cell technologies:

  • Drop-seq with antibody detection: For correlating YTHDF2 levels with transcriptome in single cells

  • Microfluidic-based RNA-protein interaction assays: For high-throughput screening of YTHDF2-RNA interactions

  • Application: Identify cell-to-cell variation in YTHDF2 activity within heterogeneous tumors

These technologies will enable more precise characterization of YTHDF2's dynamic interactions, subcellular trafficking, and functional impacts on target RNAs, advancing our understanding of its roles in cancer biology and potentially revealing new therapeutic strategies targeting the m6A pathway .

How might YTHDF2 antibodies be used in developing therapeutic approaches for cancer?

YTHDF2 antibodies can contribute to cancer therapeutic development through these innovative approaches:

• Target validation and patient stratification:

  • Immunohistochemistry-based screening to identify YTHDF2-high patient populations

  • Development of companion diagnostic tests for patient selection

  • Predictive biomarker development for response to m6A pathway inhibitors

  • Tissue microarray analysis to correlate YTHDF2 expression with treatment outcomes

• Antibody-drug conjugates (ADCs):

  • Internalization studies using fluorescently-labeled YTHDF2 antibodies

  • Development of ADCs targeting YTHDF2 in high-expressing tumors

  • Optimization of linker chemistry and payload selection

  • Preclinical efficacy testing in patient-derived xenograft models

• Bifunctional degrader development:

  • PROTAC-like molecules incorporating YTHDF2-binding fragments

  • Antibody-based YTHDF2 degraders targeting the ubiquitin-proteasome system

  • Validation of degradation efficiency using YTHDF2 antibodies

  • Correlation of degradation with phenotypic outcomes

• Therapeutic resistance mechanisms:

  • Monitoring YTHDF2 expression changes during treatment

  • Investigating YTHDF2-mediated regulation of drug resistance genes

  • Combination therapy approaches targeting YTHDF2 and conventional treatments

  • Identification of bypass mechanisms through YTHDF1/3 upregulation

• Immune therapy enhancement:

  • Analysis of YTHDF2's impact on immune checkpoint expression

  • Development of combination approaches with immune checkpoint inhibitors

  • Monitoring tumor microenvironment changes after YTHDF2 modulation

  • CAR-T approaches incorporating YTHDF2 antibody fragments for targeting

• Delivery system development:

  • YTHDF2 antibody-conjugated nanoparticles for targeted delivery

  • Aptamer-antibody chimeras for improved tissue penetration

  • Validation of targeting efficiency using imaging techniques

  • Evaluation of biodistribution in preclinical models

In hepatocellular carcinoma research, this approach led to the development of small interference RNA-containing aptamer/liposomes targeting YTHDF2, which successfully inhibited tumor growth by reducing PD-L1 and VEGFA expression. By disrupting YTHDF2's ability to facilitate ETV5 mRNA translation, this therapeutic strategy demonstrated the potential for targeting m6A readers as a novel cancer treatment approach . Similar strategies could be developed for other cancer types, with appropriate modifications based on YTHDF2's context-specific roles.

What are the most important unanswered questions about YTHDF2 function in normal and disease states?

Critical unanswered questions about YTHDF2 function in normal and disease states include:

• Molecular mechanism specificity:

  • How does YTHDF2 achieve transcript specificity beyond m6A recognition?

  • What determines whether an m6A-modified transcript is targeted by YTHDF1, YTHDF2, or YTHDF3?

  • How do post-translational modifications of YTHDF2 regulate its activity?

  • What is the structural basis for YTHDF2's interaction with the mRNA decay machinery?

• Developmental and physiological roles:

  • How does YTHDF2 contribute to normal tissue homeostasis?

  • What are the phenotypic consequences of tissue-specific YTHDF2 knockout?

  • How does YTHDF2 function change during cellular differentiation and maturation?

  • What compensatory mechanisms exist when YTHDF2 is absent or dysfunctional?

• Context-dependent cancer functions:

  • Why does YTHDF2 exhibit tumor-suppressive properties in some cancers and oncogenic properties in others?

  • What determines the cancer-specific target repertoire of YTHDF2?

  • How does YTHDF2 interact with classical oncogenic and tumor-suppressive pathways?

  • Can YTHDF2 function be modulated specifically in cancer cells without affecting normal cells?

• Therapeutic targeting considerations:

  • Is YTHDF2 a viable direct therapeutic target, or should efforts focus on its upstream regulators or downstream effectors?

  • How can the m6A-YTHDF2 axis be targeted with high specificity?

  • What biomarkers predict response to YTHDF2-targeting strategies?

  • What resistance mechanisms might emerge from YTHDF2-targeted therapies?

• Immune system interactions:

  • How does YTHDF2 regulate immune cell function?

  • What role does YTHDF2 play in the tumor microenvironment beyond cancer cells?

  • How does YTHDF2 contribute to immune surveillance and evasion?

  • Can YTHDF2 modulation enhance immunotherapy approaches?

• Integration with other epitranscriptomic mechanisms:

  • How does YTHDF2 function coordinate with other RNA modifications?

  • What is the interplay between m6A writers, erasers, and readers in determining RNA fate?

  • How do other RNA-binding proteins compete or cooperate with YTHDF2?

  • What is the evolutionary conservation of YTHDF2 function?

Addressing these questions requires integrated approaches combining structural biology, systems biology, and translational research. The seemingly contradictory findings in lung adenocarcinoma, where YTHDF2 inhibits migration and invasion , versus hepatocellular carcinoma, where it promotes tumor growth through immune evasion , highlight the context-dependent nature of YTHDF2 function and underscore the need for cancer-specific investigations to fully understand its roles in disease pathogenesis and potential therapeutic applications.