mfsd4a Antibody

What is MFSD4A and what cellular functions does it perform?

MFSD4A (Major facilitator superfamily domain-containing protein 4A) is a transmembrane protein belonging to the major facilitator superfamily of transporters. It functions as a tumor suppressor in various cancers, particularly nasopharyngeal carcinoma (NPC). Structurally, MFSD4A contains multiple transmembrane segments predicted by topology tools including TMHMM, Phobius, and Sousi .

Functionally, MFSD4A can bind to and degrade EPH receptor A2 (EPHA2) by recruiting ring finger protein 149 (RNF149), which leads to alterations in the EPHA2-mediated PI3K-AKT-ERK1/2 pathway and inhibition of epithelial-mesenchymal transition (EMT) . Through these mechanisms, MFSD4A inhibits proliferation, invasion, and migration of cancer cells. The protein is distributed in both the nucleus and cytoplasm, though its interaction with EPHA2 and RNF149 occurs primarily in the cytoplasm .

What are the available types of MFSD4A antibodies for research applications?

Several MFSD4A antibody formats are available for research purposes:

• Unconjugated antibodies: These are purified through protein A columns and peptide affinity purification, suitable for applications like Western blotting and ELISA .

• HRP-conjugated antibodies: These antibodies are directly conjugated to horseradish peroxidase, eliminating the need for secondary antibodies in applications like ELISA .

• FITC-conjugated antibodies: These fluorescently-labeled antibodies (excitation/emission: 499/515 nm) are appropriate for immunofluorescence and flow cytometry applications .

Most commercially available MFSD4A antibodies are polyclonal, raised in rabbits against human MFSD4A, with various immunogens including:

• KLH-conjugated synthetic peptides from the central region (aa 262-290)

• Recombinant human MFSD4A protein fragments (aa 160-220)

How should researchers optimize MFSD4A antibody use in Western blot applications?

For optimal Western blot detection of MFSD4A:

• Sample preparation: Extract proteins from cells or tissues using RIPA buffer supplemented with protease inhibitors. When working with NPC cells, mechanical homogenization in a bullet blender followed by standard protein extraction protocols has been effective .

• Protein loading and separation: Load 20-40 μg total protein per lane. The calculated molecular weight of MFSD4A is 56.3 kDa , so use an appropriate percentage gel (10-12%) for optimal separation.

• Antibody dilution: For unconjugated antibodies, a 1:1000 dilution is recommended as a starting point . Optimal concentration should be determined empirically for each experimental system.

• Validation controls: Include positive controls (tissues known to express MFSD4A, such as normal nasopharyngeal epithelial tissues) and negative controls (tissues with low expression, like certain NPC cell lines) .

• Signal detection: Both chemiluminescence and fluorescence-based detection systems can be used, depending on the conjugation of the primary or secondary antibody.

The detection of MFSD4A in research has successfully distinguished expression levels between normal nasopharyngeal epithelial cells (higher expression) and NPC cell lines (lower expression) .

What are the optimal protocols for immunohistochemical detection of MFSD4A in clinical specimens?

For immunohistochemical (IHC) detection of MFSD4A in clinical specimens:

• Tissue preparation: Formalin-fixed, paraffin-embedded (FFPE) tissue sections (4-5 μm thickness) should be deparaffinized and rehydrated following standard protocols.

• Antigen retrieval: Heat-induced epitope retrieval in citrate buffer (pH 6.0) is recommended to unmask antigens.

• Blocking: Block endogenous peroxidase with 3% H₂O₂ and non-specific binding with 5-10% normal serum.

• Primary antibody incubation: Incubate with MFSD4A antibody at optimized dilution (typically 1:100-1:200) overnight at 4°C or for 1-2 hours at room temperature.

• Detection system: Use a polymer-HRP detection system followed by DAB chromogen development and hematoxylin counterstaining.

• Scoring system: Implement a semi-quantitative scoring system that considers both staining intensity and percentage of positive cells. For prognostic studies, patients can be classified into MFSD4A-high and MFSD4A-low groups based on these scores .

How can MFSD4A antibodies be utilized in co-immunoprecipitation (Co-IP) experiments to study protein interactions?

For effective Co-IP experiments investigating MFSD4A interactions:

• Cell lysis: Lyse cells in a non-denaturing buffer (typically containing 1% NP-40 or Triton X-100, 150 mM NaCl, 50 mM Tris pH 7.4, plus protease inhibitors) to preserve protein-protein interactions.

• Pre-clearing: Pre-clear lysates with protein A/G beads to reduce non-specific binding.

• Immunoprecipitation: Incubate cleared lysates with MFSD4A antibody (2-5 μg per mg of total protein) overnight at 4°C, followed by addition of protein A/G beads.

• Washing and elution: Wash immunoprecipitates thoroughly (at least 3-5 times) and elute bound proteins.

• Detection: Analyze precipitated proteins by Western blotting with antibodies against suspected interaction partners.

This approach has successfully demonstrated that MFSD4A binds to EPHA2 and RNF149. When Flag-tagged MFSD4A was used to pull down GFP-tagged EPHA2 and HA-tagged RNF149, the interaction was confirmed. Similarly, GFP-tagged EPHA2 pulled down Flag-tagged MFSD4A and HA-RNF149, and HA-tagged RNF149 pulled down Flag-tagged MFSD4A and GFP-tagged EPHA2 .

How does DNA methylation affect MFSD4A expression in nasopharyngeal carcinoma?

DNA methylation plays a critical role in regulating MFSD4A expression in nasopharyngeal carcinoma (NPC):

• Hypermethylation pattern: The promoter region of MFSD4A is hypermethylated in NPC cells compared to normal nasopharyngeal epithelial cells. This can be assessed using bisulfite pyrosequencing methods .

• Relationship to expression: This hypermethylation leads to decreased MFSD4A expression at both mRNA and protein levels. Quantitative real-time PCR and Western blotting have confirmed lower expression of MFSD4A in NPC cell lines and tissues compared to normal controls .

• Experimental verification: Treatment with 5-aza-2'-deoxycytidine (DAC), an inhibitor of DNA methyltransferase, reduces MFSD4A methylation and increases its expression in NPC cell lines. This provides experimental confirmation of the causal relationship between methylation and expression .

• Clinical correlation: Hypermethylation of MFSD4A correlates with reduced expression in clinical specimens from NPC patients, establishing this as a clinically relevant mechanism .

This epigenetic silencing of MFSD4A represents an important molecular mechanism in NPC pathogenesis and offers potential for both diagnostic applications and therapeutic targeting.

What is the mechanistic pathway by which MFSD4A functions as a tumor suppressor in nasopharyngeal carcinoma?

MFSD4A exerts its tumor-suppressive effects through a complex molecular mechanism:

• EPHA2 degradation pathway: MFSD4A specifically binds to EPHA2 (EPH receptor A2) and recruits RNF149 (ring finger protein 149), an E3 ubiquitin ligase. This protein complex promotes ubiquitination and subsequent degradation of EPHA2 .

• Downstream signaling effects: Degradation of EPHA2 leads to inhibition of the PI3K-AKT-ERK1/2 signaling pathway. Specifically:

  • Reduced phosphorylation of PI3K, AKT, and ERK1/2

  • Suppression of epithelial-mesenchymal transition (EMT)

  • Altered expression of EMT markers

• Cellular phenotype impact: These molecular changes result in:

  • Decreased proliferation of NPC cells

  • Reduced migration and invasion capabilities

  • Increased apoptosis

  • Suppression of tumor growth and metastasis in vivo

• Experimental verification: The mechanism has been validated through multiple approaches including mass spectrometry, co-immunoprecipitation, and immunofluorescence assays. These techniques have confirmed the colocalization and physical interaction between MFSD4A, EPHA2, and RNF149, primarily in the cytoplasm .

This detailed mechanistic understanding provides potential targets for therapeutic intervention in NPC treatment.

How does the prognostic value of MFSD4A compare with EPHA2 in nasopharyngeal carcinoma patients?

The comparative prognostic value of MFSD4A and EPHA2 in nasopharyngeal carcinoma patients reveals an inverse relationship:

This inverse prognostic relationship is consistent with the molecular mechanism whereby MFSD4A degrades EPHA2, thus inhibiting its oncogenic effects through the PI3K-AKT-ERK1/2 pathway.

How can MFSD4A be targeted for therapeutic development in cancer?

Based on its tumor-suppressive function, MFSD4A represents a promising therapeutic target for cancer treatment:

• Epigenetic modulation approach:

  • DNA methyltransferase inhibitors (like 5-aza-2'-deoxycytidine) could reverse hypermethylation of the MFSD4A promoter

  • This would restore MFSD4A expression in cancers where it is epigenetically silenced

  • Such approaches could be particularly relevant for nasopharyngeal carcinoma

• MFSD4A protein replacement strategies:

  • Gene therapy approaches could deliver functional MFSD4A to tumor cells

  • Targeted delivery systems would need to be optimized for specific cancer types

• EPHA2 degradation pathway exploitation:

  • Small molecules that mimic MFSD4A's ability to recruit RNF149 to EPHA2

  • Proteolysis-targeting chimeras (PROTACs) that could induce EPHA2 degradation, mimicking MFSD4A's natural function

• Combinatorial approaches:

  • MFSD4A-based therapies could potentially synergize with PI3K-AKT-ERK1/2 pathway inhibitors

  • Such combinations might overcome resistance mechanisms observed with single-agent approaches

Research has shown that when MFSD4A is experimentally overexpressed in NPC cells, it results in smaller tumors and fewer metastases in animal models, validating its potential as a therapeutic target .

What methodological considerations are important when studying the role of MFSD4A-AS1 in lymphatic metastasis?

MFSD4A-AS1, the antisense long non-coding RNA corresponding to MFSD4A, requires specific methodological approaches when studying its role in lymphatic metastasis:

• Expression analysis considerations:

  • When comparing MFSD4A-AS1 expression between cancer tissues with and without lymphatic metastasis, careful patient stratification is critical

  • In papillary thyroid cancer (PTC), MFSD4A-AS1 is specifically upregulated in tissues with lymphatic metastasis, requiring precise sample categorization (TC-N1 vs. TC-N0)

• Functional assessment techniques:

  • In vitro models: Human umbilical vein endothelial cell (HUVEC) mesh formation assays provide insights into lymphangiogenic potential

  • Migration and invasion assays: Transwell systems can evaluate cancer cell motility changes induced by MFSD4A-AS1

  • In vivo models: Subcutaneous injection of cancer cells with modified MFSD4A-AS1 expression followed by analysis of lymphatic vessel density (using CD31 and PDPN markers)

• Mechanistic investigation approaches:

  • RNA immunoprecipitation assays to identify microRNA interactions

  • Luciferase reporter assays to confirm competing endogenous RNA functions

  • Analysis of VEGFA and VEGFC expression changes as downstream effectors

• Technical validation requirements:

  • Multiple cell lines to ensure reproducibility

  • Both gain- and loss-of-function experiments

  • Correlation with clinical outcomes data

Unlike MFSD4A protein which acts as a tumor suppressor, MFSD4A-AS1 promotes lymphatic metastasis by functioning as a competing endogenous RNA that sequesters miRNAs (miR-30c-2-3p, miR-145-3p and miR-139-5p), highlighting the complex and sometimes opposing roles of sense and antisense transcripts from the same genomic locus .

What techniques are most effective for studying MFSD4A expression across different tissue types?

For comprehensive analysis of MFSD4A expression across tissues:

• Quantitative RT-PCR optimization:

  • Reference gene selection is critical for cross-tissue comparison; multiple reference genes should be tested for stability across tissues

  • Primer design should account for potential transcript variants

  • For murine studies, validated primer sets have been used for Mfsd4a with the following parameters: 95°C initial denaturation (30 sec), followed by 50 cycles of 95°C (10 sec), 55-61°C (30 sec), and 72°C (30 sec)

• Western blot considerations:

  • Tissue-specific extraction protocols may be necessary to optimize protein yield

  • For brain tissues, mechanical homogenization in a bullet blender has proven effective

  • Protein loading should be normalized using housekeeping proteins stable across different tissues

• Immunohistochemistry for tissue distribution studies:

  • Antigen retrieval protocols may need tissue-specific optimization

  • Background autofluorescence varies by tissue and requires appropriate controls

  • Multiplexed staining with tissue-specific markers can help identify MFSD4A-expressing cell types

• Single-cell analysis approaches:

  • Single-cell RNA sequencing can reveal cell-type specific expression patterns

  • Tissue dissociation protocols should be optimized to maintain cellular integrity and RNA quality

Research has demonstrated that MFSD4A is expressed in both central and peripheral tissues, with expression detected in diverse organs including brainstem, cerebellum, cortex, eye, heart, hippocampus, hypothalamus, intestine, kidney, liver, lungs, olfactory bulb, ovary, spinal cord, spleen, striatum, thalamus, thymus, and uterus .

What are common pitfalls when using MFSD4A antibodies in experimental applications and how can they be addressed?

Researchers may encounter several challenges when working with MFSD4A antibodies:

• Antibody specificity issues:

  • Problem: Cross-reactivity with related proteins in the major facilitator superfamily

  • Solution: Validate antibody specificity using MFSD4A knockout/knockdown controls; consider using antibodies targeting different epitopes to confirm results

• Low signal strength in Western blots:

  • Problem: MFSD4A may be expressed at low levels in certain tissues or cell lines

  • Solution: Increase protein loading (up to 50-60 μg); optimize antibody concentration; use enhanced chemiluminescence detection systems; consider membrane protein enrichment protocols

• Background issues in immunofluorescence:

  • Problem: High background when using FITC-conjugated MFSD4A antibodies

  • Solution: Optimize blocking conditions (5-10% serum from same species as secondary antibody); increase washing stringency; reduce primary antibody concentration; use DAPI to distinguish true nuclear signal from autofluorescence

• Inconsistent immunoprecipitation results:

  • Problem: Variable efficiency in pulling down MFSD4A complexes

  • Solution: Optimize lysis conditions to preserve protein interactions; pre-clear lysates thoroughly; consider crosslinking approaches for transient interactions; use tagged MFSD4A constructs (Flag-tagged MFSD4A has shown success in Co-IP experiments)

• Sample preparation challenges:

  • Problem: MFSD4A as a transmembrane protein may be difficult to extract efficiently

  • Solution: Use detergent combinations optimized for membrane proteins; avoid excessive heating during sample preparation; consider native protein extraction for functional studies

Addressing these technical issues can significantly improve experimental outcomes when working with MFSD4A antibodies.

How can researchers differentiate between MFSD4A protein and its antisense transcript (MFSD4A-AS1) in experimental settings?

Differentiating between MFSD4A protein and its antisense transcript MFSD4A-AS1 requires careful methodological approaches:

• RNA detection specificity:

  • Primer design: Design strand-specific primers for qRT-PCR that specifically amplify either MFSD4A or MFSD4A-AS1

  • RT-PCR protocol: Use strand-specific reverse transcription with primers that target only the sense or antisense transcript

  • RNA-FISH: Employ fluorescent in situ hybridization with strand-specific probes to visualize cellular localization differences

• Protein vs. RNA detection:

  • Experimental approach: Combine immunodetection (for MFSD4A protein) with RNA detection methods (for MFSD4A-AS1)

  • Control validation: Include RNase treatment controls to eliminate RNA signal while preserving protein detection

  • Correlation analysis: Analyze whether MFSD4A protein and MFSD4A-AS1 transcript levels correlate or show inverse relationships across samples

• Functional distinction strategies:

  • Selective knockdown: Use siRNAs/shRNAs designed to target either MFSD4A mRNA or MFSD4A-AS1 specifically

  • Subcellular fractionation: Separate nuclear and cytoplasmic fractions to exploit differential localization (MFSD4A-AS1 may show different subcellular distribution than MFSD4A mRNA)

  • Context-specific expression: Leverage the finding that MFSD4A protein is downregulated in NPC while MFSD4A-AS1 is upregulated in PTC with lymphatic metastasis

• Interpretative considerations:

  • Remember that they may have opposing functions - MFSD4A protein acts as a tumor suppressor in NPC, while MFSD4A-AS1 promotes lymphatic metastasis in PTC

  • Consider analyzing both simultaneously to understand potential regulatory relationships

This methodological approach accounts for the distinct molecular nature and potentially opposing functions of the protein-coding gene and its antisense transcript.

How might single-cell analysis techniques advance our understanding of MFSD4A function across different cell populations?

Single-cell analysis technologies offer powerful approaches to understand MFSD4A biology in heterogeneous tissues:

• Single-cell RNA sequencing applications:

  • Cell type-specific expression patterns: Identify which specific cell types within tumors or normal tissues express MFSD4A at highest levels

  • Expression correlation networks: Discover co-expressed genes that may function with MFSD4A in specific cell populations

  • Trajectory analysis: Map changes in MFSD4A expression during cellular differentiation or malignant transformation

• Single-cell protein analysis approaches:

  • Mass cytometry (CyTOF): Quantify MFSD4A protein levels alongside other cancer markers at single-cell resolution

  • Imaging mass cytometry: Visualize MFSD4A distribution in tissue sections with subcellular resolution while preserving spatial context

  • Single-cell Western blotting: Quantify MFSD4A protein levels in individual cells to capture cell-to-cell variability

• Integrative multi-omics strategies:

  • CITE-seq: Simultaneously profile surface proteins and transcriptomes to correlate MFSD4A mRNA with protein expression

  • Spatial transcriptomics: Map MFSD4A expression patterns within the tumor microenvironment and at invasion fronts

• Functional screening at single-cell level:

  • CRISPR screening with single-cell readouts: Assess how MFSD4A perturbation affects diverse cell populations differently

  • Single-cell secretome analysis: Examine how MFSD4A affects secretory profiles of individual cells

These approaches could reveal previously unappreciated heterogeneity in MFSD4A expression and function across different cell types within tumors, potentially explaining variable responses to therapies targeting pathways affected by MFSD4A.

What are the implications of MFSD4A structural predictions for developing targeted therapeutic approaches?

Structural predictions of MFSD4A provide valuable insights for therapeutic development:

• Transmembrane topology-based drug design:

  • MFSD4A contains multiple predicted transmembrane segments, with topology tools like TMHMM, Phobius, and Sousi identifying its membrane-spanning domains

  • These predictions suggest potential binding pockets for small molecule development

  • The tertiary structure model built using the MFS lactose permease from E. coli as a template provides a framework for structure-based drug design

• Functional domain targeting strategies:

  • The characteristic cytoplasmic loop between transmembrane segments 6 and 7, a conserved MFS motif, could be targeted to modulate MFSD4A function

  • Peptide mimetics designed to interact with specific domains might alter MFSD4A activity or protein-protein interactions

• Protein-protein interaction interface approaches:

  • Structural understanding of how MFSD4A interacts with EPHA2 and RNF149 could enable development of:

    • Stabilizers of these interactions to enhance EPHA2 degradation

    • Peptides that mimic the interaction surfaces

    • Small molecules that promote assembly of the degradation complex

• Conformation-specific targeting:

  • Like other MFS transporters, MFSD4A likely undergoes conformational changes during its functional cycle

  • Compounds that selectively bind to and stabilize specific conformational states could modulate activity

• Structure-guided antibody development:

  • Structural predictions can identify exposed epitopes ideal for therapeutic antibody development

  • Such antibodies could either block or enhance MFSD4A interactions with partners like EPHA2