MFSD4A Antibody

What is MFSD4A and what cellular functions has it been associated with?

MFSD4A (major facilitator superfamily domain containing 4A) is a protein that belongs to the major facilitator superfamily. Research indicates that MFSD4A functions as a tumor suppressor in various cancers, particularly nasopharyngeal carcinoma (NPC). The protein is expressed in normal nasopharyngeal epithelial tissues at higher levels compared to cancerous tissues .

MFSD4A is distributed in both the nucleus and cytoplasm, with significant functional interactions occurring primarily in the cytoplasmic compartment . Its reduced expression in cancer is frequently caused by hypermethylation of its promoter region, suggesting epigenetic regulation plays a crucial role in modulating its tumor-suppressive functions .

What experimental methods are most effective for detecting MFSD4A expression in research specimens?

Based on published research protocols, several complementary methods have proven effective for MFSD4A detection:

For reliable results, researchers should employ antibodies validated for specific applications. The MFSD4A polyclonal antibody has been validated for ELISA, Western blot (WB), and immunofluorescence (IF) applications . When studying clinical specimens, immunohistochemistry has been effectively used to correlate MFSD4A expression with patient prognosis .

How does DNA methylation affect MFSD4A expression and what methods can detect this relationship?

DNA methylation significantly regulates MFSD4A expression, with hypermethylation of the promoter region leading to decreased expression. This relationship has been experimentally confirmed using several approaches:

When treated with 5-aza-2ʹ-deoxycytidine (DAC), an inhibitor of DNA methyltransferase, NPC cell lines showed both reduced methylation of the MFSD4A promoter and increased expression of MFSD4A . This finding demonstrates a direct causal relationship between methylation status and gene expression.

To study this relationship, researchers have successfully employed:

• Bisulfite pyrosequencing to quantify methylation levels

• Quantitative RT-PCR to measure mRNA expression changes after demethylation treatment

• Western blotting to confirm protein level changes

• Bioinformatic analysis to identify potential methylation sites

What is the molecular mechanism by which MFSD4A inhibits cancer progression?

MFSD4A exerts its tumor-suppressive effects through a complex mechanistic pathway involving protein-protein interactions and targeted protein degradation. Research has revealed that:

MFSD4A specifically binds to EPH receptor A2 (EPHA2) and recruits ring finger protein 149 (RNF149), an E3 ubiquitin ligase . This interaction promotes EPHA2 ubiquitination and subsequent degradation, as confirmed by co-immunoprecipitation, mass spectrometry, and immunofluorescence assays .

The degradation of EPHA2 leads to suppression of the downstream PI3K-AKT-ERK1/2 signaling pathway and inhibition of epithelial-mesenchymal transition (EMT) . These changes ultimately result in reduced cancer cell proliferation, invasion, and migration.

Experimental validation of this mechanism included:

• Co-IP assays showing MFSD4A pulled down both EPHA2 and RNF149

• Immunofluorescence demonstrating colocalization of these proteins

• Ubiquitination assays confirming increased EPHA2 ubiquitination when MFSD4A was overexpressed

What experimental approaches can effectively demonstrate the functional impact of MFSD4A in cancer cells?

Multiple complementary approaches have proven effective for studying MFSD4A's functional impact:

In vitro functional assays:

• Cell Counting Kit-8 (CCK-8) assays have demonstrated that NPC cells with downregulated MFSD4A showed increased proliferation, while upregulation of MFSD4A resulted in decreased proliferation .

• Plate clone formation assays revealed similar results, with MFSD4A downregulation increasing clone numbers and upregulation decreasing them .

• Transwell assays showed that MFSD4A expression levels inversely correlated with migration and invasion abilities of NPC cells .

• Flow cytometry can evaluate apoptosis rates in response to MFSD4A expression changes.

In vivo validation:

• Animal experiments with implanted tumors showed that MFSD4A overexpression resulted in smaller and lighter tumors.

• Lung metastasis models demonstrated reduced metastatic potential with increased MFSD4A expression .

How can researchers effectively manipulate MFSD4A expression for experimental studies?

Based on published methodologies, researchers have successfully modulated MFSD4A expression using several approaches:

For effective experimental design, researchers should:

• Include appropriate negative controls (siRNA-vector/siNC for silencing experiments)

• Confirm expression changes at both mRNA and protein levels

• Use multiple distinct siRNA sequences to minimize off-target effects

• Consider stable cell lines for long-term experiments

How can MFSD4A expression be used for patient stratification and prognostic assessment?

MFSD4A expression levels have demonstrated significant prognostic value in clinical studies:

To implement such stratification, researchers have:

Similarly, low EPHA2 expression (the protein degraded by MFSD4A) was associated with better prognosis, which is consistent with the functional relationship between these proteins .

What technical considerations are important when using MFSD4A antibodies for immunohistochemistry in clinical specimens?

For reliable immunohistochemical analysis of MFSD4A in clinical specimens, researchers should consider:

Antibody selection and validation:

• Use antibodies specifically validated for immunohistochemistry applications

• Confirm specificity through positive and negative controls

• Consider polyclonal antibodies for better epitope recognition in fixed tissues

Protocol optimization:

• Antigen retrieval methods may need optimization for formalin-fixed paraffin-embedded (FFPE) samples

• Titrate antibody concentrations to minimize background while maintaining specific signal

• Include normal nasopharyngeal epithelial tissues as positive controls, as they express higher levels of MFSD4A compared to NPC tissues

Scoring and analysis:

• Implement standardized scoring systems for consistent quantification

• Consider both staining intensity and percentage of positive cells

• Use digital image analysis where possible to reduce subjective interpretation

What strategies can address challenges in detecting low levels of MFSD4A in cancer samples?

Detecting MFSD4A in cancer samples presents challenges due to its reduced expression through hypermethylation. Researchers can implement several strategies to optimize detection:

Signal amplification methods:

• Use highly sensitive detection systems such as tyramide signal amplification for immunohistochemistry

• Consider more sensitive antibody detection methods like chemiluminescence for Western blotting

• Employ nested PCR approaches for detecting low mRNA levels

Enrichment strategies:

• Use cell fractionation to concentrate compartments where MFSD4A is most abundant

• Consider immunoprecipitation followed by Western blotting for enrichment

Epigenetic modulation:

• Treat cell lines with demethylating agents like 5-aza-2ʹ-deoxycytidine to increase MFSD4A expression for positive control samples

• Create methylation-resistant overexpression constructs for functional studies

How can researchers effectively study the interaction between MFSD4A, EPHA2, and RNF149?

The tripartite interaction between MFSD4A, EPHA2, and RNF149 is central to understanding MFSD4A's tumor-suppressive function. Several effective approaches include:

Co-immunoprecipitation (Co-IP):

• Forward and reverse Co-IP using tagged constructs has successfully demonstrated these interactions

• Tagged proteins (Flag-tagged MFSD4A, GFP-tagged EPHA2, and HA-tagged RNF149) can be used to detect specific pull-downs

Immunofluorescence co-localization:

• Fluorescent labeling has confirmed co-localization primarily in the cytoplasm

• Advanced microscopy techniques such as FRET or proximity ligation assay can provide additional evidence of direct interaction

Functional validation:

• Ubiquitination assays showing increased EPHA2 ubiquitination with MFSD4A expression

• Silencing RNF149 to demonstrate reduced ubiquitination of EPHA2 despite MFSD4A presence

• Downstream signaling analysis (PI3K-AKT-ERK1/2 pathway) to confirm functional consequences of these interactions

What are the potential therapeutic implications of targeting the MFSD4A pathway in cancer?

MFSD4A has been identified as a "promising potential therapeutic target for NPC" . Several approaches warrant further investigation:

Epigenetic therapy:

• Demethylating agents to restore MFSD4A expression in cancers where it is silenced by hypermethylation

• Combination approaches with histone deacetylase inhibitors to enhance reactivation

Pathway modulation:

• Small molecule inhibitors targeting the PI3K-AKT-ERK1/2 pathway, which is activated when MFSD4A is downregulated

• Development of EPHA2 degraders to mimic MFSD4A's natural tumor-suppressive function

Gene therapy approaches:

• Viral vector-mediated MFSD4A delivery to restore expression in tumor cells

• CRISPR activation systems to upregulate endogenous MFSD4A expression

The research demonstrates that MFSD4A overexpression leads to smaller tumors and reduced metastases in animal models, suggesting clinical potential for these approaches .

What emerging technologies might enhance our understanding of MFSD4A's role in cancer biology?

Several cutting-edge technologies could significantly advance MFSD4A research:

Single-cell analysis:

• Single-cell RNA sequencing to identify cell populations with varying MFSD4A expression within heterogeneous tumors

• Single-cell proteomics to correlate MFSD4A protein levels with other signaling components

Spatial biology approaches:

• Spatial transcriptomics to map MFSD4A expression patterns within the tumor microenvironment

• Multiplexed immunofluorescence to simultaneously visualize MFSD4A, EPHA2, RNF149, and downstream signaling markers in tissue sections

Proteomics and interactomics:

• Proximity labeling approaches (BioID, APEX) to identify additional MFSD4A interaction partners

• Quantitative ubiquitinomics to comprehensively catalog MFSD4A-dependent ubiquitination events beyond EPHA2

These technologies could reveal new functions and regulatory mechanisms of MFSD4A beyond its established role in EPHA2 degradation and NPC suppression.