DFNA5 Antibody

What is DFNA5 and what are the primary applications for DFNA5 antibodies?

DFNA5 (also known as GSDME or Gasdermin E) is a 496-amino acid protein belonging to the Gasdermin family with predicted cytoplasmic and membrane-associated localization . The protein is primarily expressed in cochlear tissue but is also found in placenta, brain, heart, liver, lung, and pancreas .

Primary applications for DFNA5 antibodies include:

• Western blotting (WB) to detect protein expression levels

• Immunohistochemistry (IHC) for tissue localization studies

• Immunofluorescence (IF) for subcellular localization

• Immunoprecipitation (IP) for protein interaction studies

• Flow cytometry (FCM) for cell population analysis

Most commercial DFNA5 antibodies have been validated for Western blot applications with human, mouse, and rat samples, though specific reactivity varies by product .

How should I validate a DFNA5 antibody for my specific application?

Proper validation of DFNA5 antibodies should follow these methodological steps:

• Specificity testing: Use positive controls (tissues or cell lines known to express DFNA5) and negative controls (DFNA5 knockout samples when available)

• Cross-reactivity assessment: Test the antibody against related Gasdermin family proteins

• Application-specific validation:

  • For WB: Confirm band at expected molecular weight (~59 kDa)

  • For IHC/IF: Include peptide blocking controls to confirm specificity

  • For IP: Validate with mass spectrometry of pulled-down proteins

Several studies have used DFNA5 antibodies validated in multiple cell lines, including HepG2, H1299, and T98G cell lines as demonstrated in previous publications .

What are the common challenges in DFNA5 protein detection and how can they be addressed?

DFNA5 detection presents several challenges for researchers:

• Low endogenous expression levels: Many tissues express DFNA5 at low levels, making detection difficult without enrichment

• Antibody specificity issues: Some antibodies may cross-react with other Gasdermin family members

• Post-translational modifications: DFNA5 may undergo modifications affecting antibody recognition

Methodological solutions include:

• Using concentrated samples (50-100 μg total protein) for Western blotting

• Employing signal amplification methods for IHC/IF (e.g., tyramide signal amplification)

• Using FLAG-tagged DFNA5 constructs when studying transfected systems

• Including appropriate blocking peptides to confirm specificity

How can I design experiments to study the relationship between DFNA5 and apoptosis?

DFNA5 has been implicated in programmed cell death, with evidence suggesting its N-terminal domain induces apoptosis while the C-terminal domain regulates this activity . When designing experiments to investigate this function:

• Domain-specific constructs: Create expression vectors for:

  • Full-length DFNA5

  • N-terminal domain (exons 2-7)

  • C-terminal domain (exons 8-10)

• Apoptosis detection methods:

  • Annexin V staining coupled with flow cytometry

  • TUNEL assay for DNA fragmentation

  • Caspase activity assays

  • Mitochondrial membrane potential measurements

• Timing considerations:

  • Monitor cells at multiple time points (3, 6, 9, 12, 16, and 24 hours post-transfection)

  • Note that cells may shift from apoptotic to secondary necrotic death after 16-24 hours

• Controls:

  • Use both wild-type and mutant DFNA5 (particularly mutations that cause hearing loss)

  • Include appropriate empty vector controls

Previous studies have shown that transfection with mutant DFNA5 (lacking exon 8) or the N-terminal domain alone induces significant apoptosis compared to wild-type controls .

What experimental approaches can identify DFNA5 protein interactions and modifications?

To investigate DFNA5 protein interactions and modifications, researchers should consider:

• Protein-protein interaction studies:

  • Co-immunoprecipitation with DFNA5 antibodies

  • Proximity labeling methods like TurboID to identify transient interactions

  • Yeast two-hybrid screening for novel binding partners

• Post-translational modification analysis:

  • Phosphorylation site mapping via mass spectrometry

  • Ubiquitination assays to assess protein stability

  • Glycosylation detection using glycosidase treatment

• Subcellular localization studies:

  • Co-localization with organelle markers (e.g., ER, mitochondria)

  • Live-cell imaging with fluorescently tagged constructs

  • Fractionation experiments followed by Western blotting

Previous research has identified interactions between DFNA5 and multiple immune-related proteins, including IFIT3, IRAK1, TAB1, and IFNGR1 using TurboID proximity labeling .

How can I assess the role of DFNA5 in immune cell regulation and exhaustion?

DFNA5 has been implicated in immune cell regulation, particularly in tumor microenvironments. To investigate this function:

• Correlation analysis with immune markers:

  • Evaluate associations between DFNA5 expression and immune cell markers in public databases (GEPIA, TIMER)

  • Analyze markers for specific cell populations: CD8+ T cells, Tregs, TAMs, M1/M2 macrophages

• Co-expression studies:

  • Assess relationship between DFNA5 and immune checkpoint molecules (PDCD1/PD-1, CD274/PD-L1)

  • Use increasing concentrations of Flag-DFNA5 plasmid co-transfected with PDCD1-3xmyc or CD274-3xmyc

• Functional assays:

  • T cell exhaustion assays following DFNA5 manipulation

  • Macrophage polarization experiments

  • Cytokine production measurements

Research has shown strong correlations between DFNA5 expression and markers of M2 macrophages (CD163, VSIG4, MS4A4A) and T cell exhaustion (PDCD1, CTLA4, LAG3, TIM-3) in colon, liver, and lung cancers .

How does DFNA5 function differ in cancer versus hearing loss contexts?

DFNA5 exhibits context-dependent functions that researchers should consider when designing experiments:

In Hearing Loss:

• Mutations causing exon 8 skipping lead to a truncated protein that induces inappropriate cell death

• All identified hearing loss mutations result in the same functional consequence despite different genomic locations

• DFNA5-associated hearing loss is progressive and nonsyndromic

In Cancer:

• DFNA5 appears to function as a tumor suppressor

• Epigenetic silencing through methylation occurs in 52-65% of gastric, colorectal, and breast tumors

• DFNA5 is induced by p53 through a binding site in intron 1

• Forced expression decreases cell growth and colony formation in cancer cell lines

Methodologically, researchers should:

• Use appropriate cellular models (cochlear cells for hearing loss, cancer cell lines for tumor studies)

• Consider different readouts (auditory function versus tumor growth metrics)

• Evaluate both wild-type and mutant DFNA5 in parallel

What techniques can identify DFNA5 methylation status in cancer samples?

DFNA5 is epigenetically regulated in multiple cancers through promoter methylation. To assess methylation status:

• Bisulfite sequencing:

  • Treat DNA with bisulfite to convert unmethylated cytosines to uracil

  • Sequence the region to identify methylated CpG sites

• Methylation-specific PCR (MSP):

  • Design primers specific to methylated and unmethylated states

  • Compare amplification patterns between primers

• Pyrosequencing:

  • Quantify methylation at individual CpG sites

  • Obtain precise methylation percentages

• Chromatin immunoprecipitation (ChIP):

  • Use antibodies targeting methylated DNA

  • Combine with p53 ChIP to assess relationship between methylation and p53 binding

Studies have shown DFNA5 promoter methylation in 52-65% of primary tumors, correlating with decreased expression and increased tumor aggressiveness .

How can I investigate the relationship between DFNA5 and p53 in cellular stress responses?

The relationship between DFNA5 and p53 is an important area of investigation:

• Chromatin immunoprecipitation (ChIP):

  • Use p53 antibodies to precipitate p53-bound DNA

  • Amplify DFNA5 regulatory regions to detect binding

  • Test different stress conditions (DNA damage, oxidative stress)

• Reporter assays:

  • Create luciferase constructs containing DFNA5 promoter regions

  • Test activation following p53 overexpression or activation

• Expression analysis:

  • Monitor DFNA5 expression following p53 activation with various stimuli

  • Compare responses in p53-wild-type versus p53-mutant or null cells

• Functional studies:

  • Assess DFNA5 contribution to p53-mediated cellular outcomes

  • Use DFNA5 knockdown in combination with p53 activation

Previous research demonstrated DFNA5 induction following p53 activation, with ChIP confirming p53 binding to the DFNA5 gene, suggesting DFNA5 plays a role in p53-regulated responses to genotoxic stress .

What are the optimal conditions for using DFNA5 antibodies in Western blotting?

For optimal Western blot results with DFNA5 antibodies:

• Sample preparation:

  • Use RIPA lysis buffer with protease inhibitors

  • Load 20-50 μg total protein per lane

  • Include positive controls (e.g., HeLa, NIH-3T3, or H9C2 cell lysates)

• Electrophoresis conditions:

  • Use 10% SDS-PAGE gels

  • Expect DFNA5 to appear at approximately 59 kDa

• Transfer and blocking:

  • Transfer to PVDF membrane

  • Block with 5% nonfat milk in TBST

• Antibody dilutions:

  • Primary antibody: 1:500-1:1000 dilution

  • Incubate overnight at 4°C

  • Secondary antibody: HRP-conjugated, 1:5000 dilution, 1 hour at room temperature

• Detection methods:

  • Enhanced chemiluminescence (ECL) systems

  • Exposure times may need optimization depending on expression levels

How can I troubleshoot non-specific binding or weak signals when using DFNA5 antibodies?

When encountering issues with DFNA5 antibody performance:

• For non-specific binding:

  • Increase blocking time (2-3 hours)

  • Use alternative blocking agents (5% BSA instead of milk)

  • Increase washing steps (5 washes × 5 minutes each)

  • Try a different antibody targeting a different epitope

  • Consider using monoclonal antibodies for higher specificity

• For weak signals:

  • Increase protein loading (50-100 μg)

  • Reduce antibody dilution (1:250-1:500)

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

  • Use signal enhancement systems (e.g., biotin-streptavidin amplification)

  • Implement antigen retrieval for IHC/IF applications

• Validation approaches:

  • Use DFNA5 knockout/knockdown samples as negative controls

  • Include blocking peptides specific to your antibody

  • Compare results across multiple antibodies targeting different epitopes

What considerations are important when designing DFNA5 knockout or overexpression experiments?

When manipulating DFNA5 expression experimentally:

• For DFNA5 knockdown/knockout:

  • Consider both siRNA and CRISPR/Cas9 approaches

  • Design multiple targeting sequences to minimize off-target effects

  • Include scrambled/non-targeting controls

  • Validate knockout efficiency at both mRNA and protein levels

  • Be aware that complete knockout may affect cell viability due to DFNA5's role in cell death

• For DFNA5 overexpression:

  • Use inducible expression systems to control timing and expression levels

  • Include both wild-type and mutant constructs for comparison

  • Consider domain-specific constructs to dissect functional regions

  • Monitor cell viability closely, as overexpression may induce apoptosis

  • Use epitope tags (FLAG, Myc) when antibody detection is challenging

• Functional validation:

  • Colony formation assays to assess growth effects

  • Apoptosis assays (Annexin V, TUNEL)

  • Verify subcellular localization using microscopy

The choice between transient and stable expression should be guided by experimental goals, noting that stable DFNA5 expression may select for resistant cells due to its apoptosis-inducing capacity.

How should I interpret contradictory data between DFNA5 expression levels and functional outcomes?

Researchers often encounter seemingly contradictory results when studying DFNA5. Consider these methodological approaches to interpretation:

• Context dependency:

  • Cell type-specific effects (cochlear vs. cancer cells)

  • Wild-type vs. mutant protein effects

  • Domain-specific activities (N-terminal vs. C-terminal)

• Expression level considerations:

  • Physiological vs. overexpression levels

  • Acute vs. chronic expression changes

  • Subcellular localization differences

• Interaction with signaling pathways:

  • p53 status of the experimental system

  • MAP kinase pathway activation state

  • Mitochondrial and ER function

• Technical factors:

  • Antibody epitope accessibility in different contexts

  • Protein modification status affecting detection

  • Timing of measurements relative to cell death induction

Studies have shown that while DFNA5 can induce apoptosis, its effects vary considerably depending on cellular context and experimental conditions .

What analytical approaches can differentiate the roles of DFNA5 in immune regulation versus cell death?

DFNA5 functions in both programmed cell death and immune regulation. To distinguish these roles:

• Differential gene expression analysis:

  • Compare transcriptional profiles following DFNA5 manipulation

  • Use pathway enrichment tools to identify activated processes

  • Conduct Gene Set Enrichment Analysis (GSEA) on DFNA5 knockout models

• Temporal analysis:

  • Examine early versus late events following DFNA5 activation

  • Map the sequence of molecular events in time-course experiments

• Domain-specific manipulations:

  • Utilize constructs that separate cell death and immune regulatory functions

  • Test point mutations that affect specific interactions

• Contextual studies:

  • Compare effects in immune versus non-immune cells

  • Assess outcomes in inflammatory versus non-inflammatory conditions

Research has identified distinct sets of interacting proteins related to immune function (IFIT3, IRAK1, TAB1, IFNGR1) versus cell death regulation, suggesting separable molecular mechanisms .

What correlation exists between DFNA5 expression and immune cell markers in different cancer types?

The table below summarizes the correlation coefficients between DFNA5 expression and immune cell markers in different cancer types:

*P < 0.01, **P < 0.001, ***P < 0.0001
COAD: colon adenocarcinoma; LIHC: liver hepatocellular carcinoma; LUAD: lung adenocarcinoma

This data demonstrates strong correlations between DFNA5 expression and multiple immune cell markers, particularly in colon cancer, suggesting a significant role in immune regulation within the tumor microenvironment.

What DFNA5 antibody applications have been validated across different experimental systems?

The following table summarizes validated applications for DFNA5 antibodies based on research literature:

This comprehensive validation data helps researchers select appropriate antibodies and conditions for their specific experimental systems .

How do different domains of DFNA5 contribute to its function and localization?

The table below summarizes the functional and localization properties of different DFNA5 domains:

This domain analysis explains why mutations causing exon 8 skipping lead to hearing loss through inappropriate cell death, as they eliminate the regulatory function of Domain B that normally prevents Domain A-induced apoptosis .

What emerging techniques could advance our understanding of DFNA5 function?

Emerging methodologies that could address current knowledge gaps include:

• Single-cell analysis techniques:

  • scRNA-seq to identify cell populations affected by DFNA5

  • CyTOF for protein-level analysis in heterogeneous samples

  • Spatial transcriptomics to map DFNA5 expression in tissue context

• Advanced protein analysis:

  • Hydrogen-deuterium exchange mass spectrometry to identify conformational changes

  • Cryo-EM to resolve DFNA5 structure in different activation states

  • Optogenetic control of DFNA5 domains to study temporal dynamics

• In vivo models:

  • Conditional and tissue-specific DFNA5 knockout/knockin mice

  • CRISPR-edited animal models mimicking human mutations

  • Patient-derived organoids to study disease-specific effects

• Therapeutic targeting approaches:

  • Small molecule screens to identify DFNA5 modulators

  • Domain-specific inhibitors to selectively block apoptotic function

  • Strategies to restore DFNA5 expression in methylated tumors