MTHFD2 Antibody

What is MTHFD2 and why is it an important research target?

MTHFD2 (Methylenetetrahydrofolate Dehydrogenase 2) is a bifunctional enzyme involved in mitochondrial one-carbon metabolism that has emerged as a critical metabolic checkpoint in various cellular processes. It plays essential roles in:

• Regulating de novo purine synthesis in activated T cells, affecting proliferation and inflammatory cytokine production

• Promoting DNA replication and maintaining genomic stability in cancer cells

• Controlling cell cycle progression, particularly through S-phase regulation

• Mediating osteoclastogenesis and bone remodeling in inflammatory conditions

MTHFD2 is particularly notable as it shows high expression in embryonic tissues and multiple cancer types while maintaining low expression in normal adult tissues . Its overexpression has been documented in various inflammatory and autoimmune diseases, including rheumatoid arthritis, Crohn's disease, lupus, and multiple sclerosis . This distinctive expression pattern makes MTHFD2 both an informative biomarker and a potential therapeutic target.

What types of MTHFD2 antibodies are available for research and how should I select the appropriate one?

Several types of MTHFD2 antibodies are commercially available with distinct properties and applications:

Selection criteria should include:

• Intended application: Verify the antibody has been validated for your specific application (WB, IHC, IF, etc.)

• Species reactivity: Confirm compatibility with your experimental model (human, mouse, rat)

• Epitope location: Consider N-terminal vs. C-terminal targeting depending on your research questions

• Validation data: Examine knockout validation and positive controls

• Clonality: Monoclonal for consistent detection of specific epitopes; polyclonal for robust detection of the protein under varying conditions

For studies examining MTHFD2 in both nuclear and mitochondrial compartments, select antibodies validated for subcellular localization studies .

What are the optimal conditions for using MTHFD2 antibodies in Western blotting?

Successful Western blotting for MTHFD2 requires optimization of several parameters:

Sample preparation:

• MTHFD2 is predominantly expressed in mitochondria, but has also been detected in the nucleus

• Use appropriate fractionation methods when studying subcellular localization

• For total protein, standard RIPA buffer supplementation with protease inhibitors is generally sufficient

Running conditions:

• MTHFD2 has a calculated molecular weight of approximately 38 kDa

• 10-12% polyacrylamide gels provide optimal resolution

Antibody dilutions and controls:

• Primary antibody: Typically 1:500-1:1000 dilution (e.g., ab307428 at 1:1000)

• Secondary antibody: HRP-conjugated at 1:5000-1:20000

• Always include a positive control (e.g., HEK293T wild-type lysate)

• Include a negative control (e.g., MTHFD2 knockout lysate) to confirm specificity

Validation approach:
To confirm MTHFD2 antibody specificity, compare bands between:

• Wild-type samples (should show band at ~38 kDa)

• MTHFD2 knockout samples (band should be absent)

• Protein loading control (e.g., beta-tubulin) to normalize expression

A study examining MTHFD2 interacting proteins used immunoprecipitation followed by Western blotting with two distinct MTHFD2 antibodies to confirm specificity (Proteintech 12270-1AP and Genetex N3C3) .

How can MTHFD2 antibodies be applied for immunohistochemistry in cancer and inflammatory disease research?

MTHFD2 immunohistochemistry (IHC) has proven valuable for studying expression patterns in diseased tissues, particularly in cancer and inflammatory conditions.

Tissue preparation and antigen retrieval:

• Formalin-fixed paraffin-embedded (FFPE) tissues are commonly used

• Heat-induced epitope retrieval using citrate buffer (pH 6.0) or EDTA buffer (pH 9.0)

• Blockade of endogenous peroxidase activity is critical for reducing background

Antibody selection and controls:

• Monoclonal antibodies (e.g., EPR26938-20 , 4G7-2G3 ) have been validated for IHC-P

• Include positive control tissues with known MTHFD2 expression (e.g., embryonic tissues, specific cancer samples)

• Include negative controls using isotype-matched antibodies

Interpretation considerations:

• MTHFD2 shows predominantly mitochondrial staining pattern but can also appear in nuclei

• In cancer samples, evaluate both staining intensity and proportion of positive cells

• Compare expression between tumor tissue and adjacent normal tissue

• In inflammatory conditions like rheumatoid arthritis, focus on expression in immune cell infiltrates and osteoclasts

Research has shown that MTHFD2 expression correlates with poor prognosis in multiple cancer types, including breast cancer and colorectal cancer . Quantitative scoring systems incorporating both staining intensity and percentage of positive cells can provide clinically relevant expression metrics.

What approaches are recommended for visualizing MTHFD2 subcellular localization?

MTHFD2 has been detected in both mitochondria and the nucleus , requiring specialized techniques to accurately visualize its distribution:

Immunofluorescence optimization:

• Fixation: 4% paraformaldehyde or 80% methanol (5 min)

• Permeabilization: 0.1-0.3% Triton X-100 or 0.1% PBS-Tween (20 min)

• Primary antibody dilution: typically 1:100-1:500

• Counter-staining recommendations:

  • Mitochondrial markers: MitoTracker, TOMM20, or COX IV

  • Nuclear markers: DAPI or Hoechst

  • ER markers: Calnexin or PDI (if assessing potential association with mitochondria-associated membranes)

Co-localization analysis:

• Capture high-resolution z-stack images

• Apply deconvolution algorithms

• Calculate co-localization metrics (Pearson's coefficient, Manders' overlap coefficient)

• Perform quantitative analysis across multiple cells/fields

Research by Nilsson et al. demonstrated that MTHFD2 physically interacts with nuclear proteins involved in RNA metabolism and translation, suggesting important non-canonical functions beyond its metabolic role . This highlights the importance of carefully examining both mitochondrial and nuclear compartments when studying MTHFD2 localization.

How can MTHFD2 antibodies be used to investigate protein-protein interactions and non-metabolic functions?

Recent research has revealed MTHFD2 interactions with numerous non-metabolic proteins, suggesting functions beyond its enzymatic role in one-carbon metabolism:

Co-immunoprecipitation (Co-IP) approaches:

• Use two distinct MTHFD2 antibodies to confirm specificity (e.g., Proteintech 12270-1AP and Genetex N3C3)

• Include both wild-type and MTHFD2 knockout cells as controls

• Analyze immunoprecipitated proteins by mass spectrometry

• Validate key interactions with reverse Co-IP

Key protein interaction findings:
A comprehensive Co-IP study identified 29 high-confidence MTHFD2-interacting proteins (D2PPI), including:

• RNA binding proteins (hnRNP family members, splicing factors)

• Heat shock proteins (HSPA8, HSPA9, HSPB1, HSPD1)

• Ribosomal proteins (RPS13, RPS3A, RPS5, RPS8)

• Histones (H3 variants)

• DNA repair proteins (XRCC6, RPA1)

Functional implications:

• MTHFD2 may participate in RNA processing and metabolism

• Potential role in stress response pathways

• Possible involvement in chromatin regulation and DNA repair

• Contribution to ribosome function and translation

These findings suggest investigating MTHFD2 beyond its metabolic function when designing experiments. For example, researchers should consider how MTHFD2 inhibition might affect transcription, translation, or DNA repair mechanisms in addition to metabolic pathways.

What methodological approaches are recommended for studying the role of MTHFD2 in T cell activation and immune function?

MTHFD2 has emerged as a key metabolic checkpoint in T cell function, with significant implications for inflammatory and autoimmune diseases . When investigating its role in immune cells:

Flow cytometry protocols:

• Surface staining for T cell subset markers (CD4, CD8, etc.)

• Fixation and permeabilization for intracellular MTHFD2 staining

• Co-staining with activation markers (CD25, CD69)

• Analysis gates should include:

  • Live/dead discrimination

  • Single cell selection

  • Lineage markers

  • MTHFD2 expression levels

Experimental designs:

• Compare MTHFD2 expression before and after T cell activation (5, 24, and 48-hour timepoints)

• Correlate MTHFD2 expression with effector vs. regulatory phenotypes

• Assess impact of MTHFD2 inhibition on:

  • Proliferation (CFSE dilution)

  • Cytokine production (IFN-γ, IL-17, etc.)

  • FoxP3 expression in Th17 and Treg populations

Mechanistic investigations:

• Measure changes in purine metabolites after MTHFD2 modulation

• Assess mTORC1 signaling (phospho-S6, phospho-4EBP1)

• Evaluate DNA/histone methylation patterns

Research has shown that MTHFD2 deficiency impairs effector T cell proliferation and function while promoting regulatory T cell differentiation . MTHFD2 inhibition protected against multiple inflammatory disease models, highlighting its potential as an immunomodulatory target .

How should researchers integrate MTHFD2 protein data with metabolomic analyses to understand its functional impact?

MTHFD2's enzymatic role in one-carbon metabolism necessitates integrated analysis of both protein expression and metabolite changes:

Integrated experimental approach:

• Modulate MTHFD2 expression/activity (knockdown, knockout, or inhibitor treatment)

• Perform Western blotting to confirm protein reduction

• Conduct targeted metabolomics focusing on:

  • Purine intermediates and end products

  • Folate metabolites

  • Thymidine and related pyrimidines

  • SAM/SAH ratio (methylation indicator)

• Correlate metabolite changes with functional outcomes

Key metabolic pathways to monitor:

• De novo purine synthesis pathway

• Thymidylate synthesis pathway

• Folate cycle intermediates

• Methionine cycle components

Interpretation framework:
Research has shown that MTHFD2 inhibition leads to several key metabolic alterations:

• Depletion of purine pools

• Accumulation of purine biosynthetic intermediates (e.g., AICAR)

• Decreased thymidine production

• Misincorporation of uracil into DNA

These metabolic changes correlate with functional outcomes including:

• Reduced mTORC1 signaling

• Altered DNA and histone methylation

• Replication stress and DNA damage

• S-phase cell cycle arrest

When designing metabolomics experiments, researchers should include appropriate time points to capture both immediate metabolic shifts and downstream pathway adaptations following MTHFD2 modulation.

What considerations are important when using MTHFD2 antibodies in cancer research?

MTHFD2 is consistently overexpressed in various cancers and has emerged as both a biomarker and therapeutic target . Key considerations for cancer research include:

Expression analysis approaches:

• Compare MTHFD2 levels between matched tumor and normal tissues

• Correlate expression with clinical outcomes and pathological features

• Examine association with cancer stem cell markers

Functional studies:

• Assess impact of MTHFD2 inhibition on:

  • Cell proliferation and viability

  • Cell cycle progression (particularly S-phase)

  • DNA replication and genomic stability

  • Resistance to therapeutic agents

Mechanistic investigation recommendations:

• Study replication stress markers (γH2AX, phospho-RPA, phospho-Chk1)

• Examine mitochondrial function (membrane potential, ATP production)

• Assess oxidative stress parameters

• Analyze nucleotide pools and DNA damage markers

Research has shown that MTHFD2 inhibition in cancer cells leads to:

• Reduced replication fork speed

• Progressive accumulation of cells in S phase

• Increased DNA damage (γH2AX positive cells)

• Activation of the ATR-mediated DNA damage response pathway

• Induction of apoptosis through caspase activation

These findings demonstrate that MTHFD2 antibodies can be valuable tools for investigating both the expression and functional consequences of this enzyme in cancer models.

How can researchers effectively use MTHFD2 antibodies to study inflammatory and autoimmune conditions?

MTHFD2 is consistently overexpressed across multiple inflammatory and autoimmune diseases , making it a valuable target for investigation:

Sample selection and preparation:

• Compare MTHFD2 expression between:

  • Healthy donors vs. patients with active disease

  • Patients before and after therapeutic intervention

  • Different disease subtypes

Cell types of interest:

• CD14+ monocytes from peripheral blood

• Tissue-infiltrating macrophages

• CD4+ T cell subsets (particularly Th17 and Treg populations)

• Osteoclast precursors (in rheumatoid arthritis)

Application-specific recommendations:
For flow cytometry:

• Include markers to identify specific immune cell populations

• Add functional markers (cytokines, activation markers)

• Consider fixation protocols that preserve both surface antigens and intracellular MTHFD2

For tissue immunohistochemistry:

• Examine MTHFD2 expression at sites of inflammation

• Correlate with markers of tissue damage and immune infiltration

Research findings:
Studies have demonstrated that:

• MTHFD2 is upregulated in CD4+ T cells during activation and at inflammation sites

• MTHFD2 prevents aberrant upregulation of FoxP3 in Th17 cells

• In rheumatoid arthritis, MTHFD2 promotes osteoclastogenesis and bone loss through effects on oxidative phosphorylation

• MTHFD2 inhibition shows therapeutic potential in multiple inflammatory disease models, including experimental autoimmune encephalomyelitis and inflammatory bowel disease

What are the emerging applications of MTHFD2 antibodies in inhibitor development and therapeutic research?

MTHFD2 has emerged as a promising therapeutic target, with several inhibitor classes now in development :

Inhibitor characterization approaches:

• Use MTHFD2 antibodies to confirm target engagement

• Perform immunoprecipitation to assess inhibitor effects on protein-protein interactions

• Monitor post-translational modifications that may affect inhibitor binding

• Examine changes in subcellular localization following inhibitor treatment

Inhibitor classes and mechanisms:
Recent research has identified several MTHFD2 inhibitor classes:

• Tricyclic coumarin derivatives:

  • Compound DS44960156 (IC₅₀ = 1.6 μM)

  • Compound DS18561882 (IC₅₀ = 0.0063 μM)

• Diaminopyrimidine-based inhibitors:

  • TH7299 (IC₅₀ = 254 nM)

  • TH9028 (IC₅₀ = 11 nM)

  • TH9619 (IC₅₀ = 47 nM)

Experimental design for inhibitor assessment:

• Confirm inhibitor selectivity (MTHFD2 vs. MTHFD1/MTHFD2L)

• Assess cellular uptake and target engagement

• Monitor metabolic consequences:

  • Purine and pyrimidine pools

  • Folate cycle intermediates

• Measure functional outcomes:

  • Replication stress markers

  • Cell cycle progression

  • Apoptosis induction

Therapeutic applications:
Current research indicates potential applications in:

• Cancer therapy, particularly acute myeloid leukemia

• Inflammatory and autoimmune diseases

• Rheumatoid arthritis and bone disorders

MTHFD2 inhibitors have demonstrated a wide therapeutic window (four orders of magnitude) between cancer cells and non-tumorigenic cells, highlighting their potential clinical utility .

What are common pitfalls in MTHFD2 antibody-based experiments and how can they be addressed?

Researchers working with MTHFD2 antibodies should be aware of several technical challenges:

Specificity concerns:

• Cross-reactivity with related enzymes (MTHFD1, MTHFD2L)

• Non-specific binding in certain tissues

• Batch-to-batch variability (especially with polyclonal antibodies)

Mitigation strategies:

• Use MTHFD2 knockout samples as negative controls

• Employ two distinct antibodies targeting different epitopes

• Include appropriate blocking controls

• Consider recombinant monoclonal antibodies for consistent results

Application-specific issues:
For Western blotting:

• False bands at unexpected molecular weights

• Variable expression depending on cell growth conditions

For immunostaining:

• High background in mitochondria-rich tissues

• Fixation-dependent epitope accessibility

• Dual localization (mitochondrial and nuclear) complicating interpretation

Solution approaches:

• Optimize antibody dilutions systematically

• Test multiple fixation protocols

• Include subcellular fractionation controls

• Use super-resolution microscopy for localization studies

Two studies successfully addressed specificity concerns by:

• Using both wildtype and MTHFD2 knockout HEK293T cells

• Employing two distinct MTHFD2 antibodies to confirm protein identity

How can researchers reconcile conflicting data on MTHFD2 subcellular localization and non-canonical functions?

The literature contains some contradictory findings regarding MTHFD2 localization and functions:

Sources of potential discrepancies:

• Different antibodies detecting distinct protein conformations

• Cell type-specific localization patterns

• Condition-dependent translocation (stress, cell cycle)

• Detection of post-translationally modified forms

• Technical differences in fixation and permeabilization

Experimental approach to resolve conflicting data:

• Complementary techniques: Combine antibody-based detection with:

  • Subcellular fractionation followed by Western blotting

  • GFP/RFP-tagged MTHFD2 live imaging

  • Proximity ligation assays to confirm protein interactions

  • Mass spectrometry-based proteomic analysis of subcellular fractions

• Control experiments:

  • Use multiple validated antibodies targeting different epitopes

  • Include MTHFD2 knockout cells as negative controls

  • Perform rescue experiments with MTHFD2 constructs lacking specific domains

  • Test localization under various cellular stresses and cell cycle stages

The study by Nilsson et al. demonstrated MTHFD2 interactions with nuclear proteins using co-immunoprecipitation with two distinct antibodies, providing strong evidence for nuclear functions beyond its canonical mitochondrial role . Additionally, research has shown that MTHFD2 can participate in DNA replication and genomic stability pathways, further supporting its presence and function in the nucleus .

What emerging applications of MTHFD2 antibodies show the most promise for advancing our understanding of disease mechanisms?

Several innovative applications of MTHFD2 antibodies are opening new research avenues:

Single-cell analysis technologies:

• Mass cytometry (CyTOF) incorporation of MTHFD2 in immune cell panels

• Single-cell Western blotting to assess heterogeneity in MTHFD2 expression

• Imaging mass cytometry for spatial resolution of MTHFD2 in tissues

Pathway and network mapping:

• Proximity labeling techniques (BioID, APEX) to map MTHFD2 interaction networks

• ChIP-seq applications to investigate potential chromatin interactions

• Global proteomics to identify post-translational modifications of MTHFD2

Therapeutic monitoring:

• Development of biomarker assays using validated antibody pairs

• Companion diagnostics for MTHFD2 inhibitor trials

• Monitoring immune cell metabolic reprogramming during immunotherapy

Recent research findings suggest promising directions:

• MTHFD2 expression corresponds with inflammatory phenotypes in multiple diseases

• MTHFD2 interacts with proteins involved in RNA metabolism and translation

• MTHFD2 inhibition affects DNA replication and genomic stability

These discoveries point to MTHFD2 as a multifunctional protein with roles beyond metabolism, warranting investigation as both a biomarker and therapeutic target across various disease contexts.

What methodological advances are needed to better characterize the diverse functions of MTHFD2 in different cellular compartments?

Current evidence suggests MTHFD2 has both metabolic and non-metabolic functions in different cellular locations, requiring advanced methodological approaches:

Technical limitations to overcome:

• Difficulty distinguishing metabolic vs. non-metabolic functions

• Challenges in tracking dynamic protein relocalization

• Limited understanding of post-translational modifications

• Incomplete characterization of protein interaction networks

Promising methodological approaches:

• Domain-specific antibodies:

  • Development of antibodies recognizing specific functional domains

  • Epitope-specific antibodies detecting post-translational modifications

  • Conformation-specific antibodies for active vs. inactive forms

• Live imaging techniques:

  • Split fluorescent protein complementation to visualize protein interactions

  • FRET/BRET sensors to detect MTHFD2 conformational changes

  • Optogenetic tools to control MTHFD2 localization

• Functional compartmentalization studies:

  • Domain deletion constructs to identify localization signals

  • Targeted protein degradation approaches (PROTAC, dTAG)

  • Compartment-specific inhibition strategies

Recent research has revealed that MTHFD2 physically interacts with DNA replication proteins and RNA processing factors , suggesting it functions beyond its canonical metabolic role. Additionally, studies have shown that MTHFD2 can influence histone methylation patterns , indicating potential epigenetic functions.

Development of tools to specifically study these non-canonical functions will be crucial for fully understanding MTHFD2's complex role in normal physiology and disease states.