ETV4 Antibody

What is ETV4 and why is it significant in molecular biology research?

ETV4 (ETS variant transcription factor 4) is a 484 amino acid protein with a molecular weight of approximately 54 kDa, though it's typically observed at 61-65 kDa due to post-translational modifications like phosphorylation and sumoylation . As a member of the ETS protein family, ETV4 functions as a DNA-binding transcription factor with a nuclear subcellular localization .

The significance of ETV4 in research stems from:

• Its role in embryonic development, including neurogenesis, lung branching, spermatogenesis, and limb bud formation

• Its involvement in MAPK family signaling pathways

• Its aberrant expression in multiple cancer types, where it's associated with cancer progression, metastasis, and poor prognosis

• Its potential as a prognostic biomarker and therapeutic target

ETV4 binds to the core sequence 5'[AC]GGA[AT]GT-3', specifically recognizing the PEA-3 motif (5'-AGGAAG-3') . Unlike other ETS family members primarily expressed in hematopoietic cells, ETV4 is predominantly expressed in cells of epithelial and fibroblastic origin .

What are the key applications of ETV4 antibodies in research laboratories?

ETV4 antibodies are versatile tools employed in multiple research applications:

When designing experiments, researchers should consider that ETV4 is typically detected at 54-61 kDa, though alternative splicing can produce isoforms of different molecular weights .

How do I select the appropriate ETV4 antibody for my specific experimental needs?

Selection of an appropriate ETV4 antibody should be guided by several key considerations:

1. Experimental application:

• For Western blot: Antibodies validated specifically for WB (e.g., Proteintech #10684-1-AP at 1:500-1:1000 dilution)

• For ChIP experiments: Antibodies optimized for chromatin binding (e.g., Cell Signaling #65763)

• For multiplexed applications: Consider conjugation-ready formats

2. Target species reactivity:

• Ensure cross-reactivity with your study species (human, mouse, rat, etc.)

• Some antibodies have broader reactivity profiles (e.g., antibodies reactive with human, mouse, rat, bovine)

3. Epitope recognition:

• N-terminal targeting: For detecting all possible isoforms

• C-terminal targeting: May miss some splice variants

• Middle region: May offer balanced detection

4. Clonality:

• Polyclonal: Broader epitope recognition, potentially higher sensitivity

• Monoclonal: Higher specificity, better lot-to-lot consistency

• Recombinant monoclonal: Superior lot-to-lot consistency

5. Validation data:

• Review published literature using the antibody

• Check for validation in knockout/knockdown controls

• Request additional validation data from suppliers if needed

When studying alternative splicing variants, it's critical to select antibodies that target conserved regions present in all variants of interest .

What are the best practices for optimizing Western blot protocols with ETV4 antibodies?

Optimizing Western blot protocols for ETV4 detection requires attention to several technical aspects:

Sample preparation:

• Include phosphatase inhibitors in lysis buffers to preserve post-translational modifications

• Nuclear extraction protocols are recommended as ETV4 is predominantly nuclear

• Heating samples at 95°C for 5 minutes in reducing sample buffer helps denature the protein fully

Gel electrophoresis and transfer:

• Use 8-10% SDS-PAGE gels for optimal resolution around 54-65 kDa

• Transfer to PVDF membranes (rather than nitrocellulose) for better protein retention

Antibody incubation:

• Primary antibody dilutions typically range from 1:500-1:2000

• Overnight incubation at 4°C often yields cleaner results than shorter incubations

• 5% BSA in TBST may provide lower background than milk-based blocking solutions

Detection considerations:

• Expect bands at 54-61 kDa for full-length ETV4

• Additional bands at lower molecular weights may represent splice variants

• Phosphorylated forms may appear at slightly higher molecular weights (61-65 kDa)

Positive controls:

• BxPC-3 cells and mouse heart tissue have been validated as positive controls for ETV4 expression

• VCap, LNCap, and MCF7 cell lines typically show low or undetectable ETV4 expression

If detecting multiple splice variants is important for your research, consider using antibodies targeting conserved regions or running parallel blots with antibodies recognizing different epitopes .

How can I validate the specificity of an ETV4 antibody for my research?

Thorough validation of ETV4 antibody specificity involves multiple complementary approaches:

Genetic approaches:

• siRNA/shRNA knockdown: Compare ETV4 detection in control vs. knockdown samples

• CRISPR/Cas9 knockout: The gold standard for specificity validation

• Overexpression: Test increased signal with ETV4 overexpression constructs

Biochemical validation:

• Peptide competition assays: Pre-incubation with immunizing peptide should abolish specific signal

• Molecular weight verification: ETV4 should appear at 54-61 kDa (depending on post-translational modifications)

• Cross-reactivity testing: Test against related ETS family members

Multiple applications validation:

• Concordance across techniques: Results should be consistent between WB, IHC, and IF

• Subcellular localization: ETV4 should show nuclear localization in IF/IHC

• Expected expression patterns: Compare with literature-reported expression in tissues/cell lines

Cross-antibody validation:

• Compare results using antibodies recognizing different epitopes

• Validate with both monoclonal and polyclonal antibodies when possible

For research focused on splice variants, validation should include PCR confirmation of expressed variants alongside antibody detection to confirm proper isoform recognition .

What are the optimal fixation and antigen retrieval methods for immunohistochemistry with ETV4 antibodies?

Successful immunohistochemical detection of ETV4 requires careful attention to fixation and antigen retrieval:

Fixation protocols:

• Formalin fixation: 10% neutral buffered formalin for 24-48 hours is standard for paraffin embedding

• For frozen sections: 4% paraformaldehyde for 10-15 minutes preserves antigenicity while maintaining morphology

• Avoid overfixation as it can mask ETV4 epitopes

Antigen retrieval methods:

• Heat-induced epitope retrieval (HIER) is typically most effective:

  • Citrate buffer (pH 6.0): 20 minutes at 95-100°C

  • EDTA buffer (pH 9.0): May provide better results for some ETV4 antibodies

• Pressure cooker methods often yield superior results compared to microwave or water bath methods

Protocol optimization:

• Perform a titration series (e.g., 1:20, 1:50, 1:100, 1:200) to determine optimal antibody dilution

• Include appropriate positive control tissues (e.g., certain tumor samples known to express ETV4)

• Include negative controls (primary antibody omission and ideally ETV4-negative tissues)

Signal detection systems:

• Polymer-based detection systems often provide better signal-to-noise ratio than avidin-biotin methods

• For dual immunofluorescence studies, carefully select fluorophores to avoid spectral overlap

Different ETV4 antibodies may require different optimization parameters, so preliminary testing is essential for achieving optimal staining .

How do alternative splicing variants of ETV4 affect antibody selection and experimental interpretation?

Alternative splicing of ETV4 creates multiple isoforms with distinct functional properties, necessitating careful antibody selection and data interpretation:

Known ETV4 splice variants:

• Multiple splice variants have been identified in human cancer cell lines

• Common variants include those missing exon 4 (∆4), exon 7 (∆7), and isoform X1

• The expression pattern of variants differs between cancer types and cell lines

Strategic antibody selection:

• Target conserved domains present in all isoforms of interest

• N-terminal antibodies: May detect most variants

• C-terminal antibodies: Will miss truncated variants

• Epitope-specific antibodies: Can distinguish between specific isoforms

Experimental approaches for splice variant analysis:

• Parallel Western blots using antibodies targeting different epitopes

• Correlation with RT-PCR data to confirm isoform expression

• Long-read sequencing (e.g., Oxford Nanopore) for comprehensive transcript identification

Interpretation challenges:

• Multiple bands on Western blots may represent splice variants rather than degradation products

• Quantitative differences between antibodies may reflect isoform-specific detection

• Functional differences between isoforms may affect biological interpretations

Research indicates that in prostate cancer cell lines, alternative transcripts can account for 23-74% of total ETV4 mRNA, with the ∆7 variant typically being the most abundant . This highlights the importance of considering splice variants in experimental design and interpretation.

What are the best practices for using ETV4 antibodies in ChIP and CUT&RUN experiments?

Chromatin immunoprecipitation (ChIP) and Cleavage Under Targets and Release Using Nuclease (CUT&RUN) are powerful techniques for studying ETV4's genomic binding sites:

ChIP protocol optimization:

• Crosslinking: 1% formaldehyde for 10 minutes at room temperature

• Sonication: Optimize to achieve chromatin fragments of 200-500 bp

• Antibody amount: Typically 1-5 μg per IP reaction

• Controls: Include IgG control and input samples

• Recommended antibody dilution: 1:200 for ChIP applications

CUT&RUN advantages and considerations:

• Higher sensitivity than standard ChIP

• Requires fewer cells (as few as 500,000)

• Lower background

• Recommended antibody dilution: 1:100

• Compatible with CUT&RUN Assay Kit #86652

Antibody selection criteria:

• Validation: Choose antibodies specifically validated for ChIP/CUT&RUN

• Target epitope: Ensure the epitope is accessible in the chromatin context

• Specificity: High specificity is crucial to avoid false positives

Data analysis and validation:

• Confirm binding to known ETV4 targets

• Motif analysis should reveal enrichment of the ETV4 binding motif (5'-AGGAAG-3')

• Validate key findings with orthogonal methods (e.g., reporter assays)

Recommended ETV4 antibodies:

• Cell Signaling Technology #65763 is validated for both ChIP and CUT&RUN applications

• For multiplexed ChIP-seq, consider using antibodies compatible with barcoding strategies

When analyzing ETV4 binding in cancer models, consider the potential influence of altered expression levels and splice variants on genomic occupancy patterns .

How can I effectively use ETV4 antibodies to study its role in cancer progression?

ETV4 has emerged as an important factor in cancer progression, and antibody-based approaches offer valuable insights:

Tissue microarray analysis:

• Use validated IHC protocols to assess ETV4 expression across tumor stages

• Correlate expression with clinicopathological parameters and patient outcomes

• Compare with normal adjacent tissue to establish baseline expression

Subcellular localization studies:

• Immunofluorescence can reveal altered ETV4 localization in cancer cells

• Co-localization with other factors may suggest mechanistic interactions

• Nuclear vs. cytoplasmic distribution can have prognostic significance

Functional studies with validation:

• Combine knockdown/overexpression with antibody detection to confirm manipulation

• Use antibodies to monitor changes in downstream targets after ETV4 modulation

• Evaluate changes in phosphorylation status with phospho-specific antibodies

Cancer pathway analysis:

• ETV4 is linked to TGF-β/Smad signaling - this can be studied using inhibitors like amygdalin or activators like SRI-011381 hydrochloride

• ETV4 expression correlates with immune checkpoint genes and immune cell infiltration in multiple cancers

• Study correlation with mismatch repair genes and methyltransferases

Tumor heterogeneity assessment:

• Single-cell analysis with ETV4 antibodies can reveal expression heterogeneity

• Combined RNA-seq and protein-level detection helps identify discordant regulation

• ETV4 expression is linked to tumor heterogeneity markers including TMB, tumor purity, and MSI

Research indicates that ETV4 may promote cancer metastasis by triggering transcription of ZEB1 and SNAIL1, suggesting these as important downstream targets to monitor .

What approaches can be used to study ETV4 post-translational modifications?

ETV4 undergoes several post-translational modifications that regulate its activity and stability, requiring specialized approaches:

Phosphorylation analysis:

• Phospho-specific antibodies: Currently limited commercial availability

• Phosphorylation detection: Appears as mobility shift (54 kDa → 61-65 kDa)

• Phosphatase treatment: Compare treated vs. untreated samples to confirm phosphorylation

• Mass spectrometry: For unbiased identification of phosphorylation sites

SUMOylation studies:

• Denaturing lysis conditions: Critical to preserve SUMO modifications

• Immunoprecipitation approaches: Can enrich for SUMOylated forms

• SUMO-trap technology: To capture all SUMOylated proteins including modified ETV4

• Mutation of SUMOylation sites: To assess functional consequences

Other potential modifications:

• Ubiquitination: Study protein stability and degradation pathways

• Acetylation: May affect DNA binding and transcriptional activity

• Methylation: Potential regulator of protein-protein interactions

Experimental approaches:

• Immunoprecipitation followed by modification-specific Western blotting

• Phosphorylation state-specific antibodies (when available)

• Treatment with kinase inhibitors to identify responsible signaling pathways

• MAPK pathway modulators: ETV4 is regulated by MAPK signaling

When analyzing ETV4 by Western blot, researchers should be aware that post-translational modifications cause the protein to migrate at a higher apparent molecular weight (61-65 kDa) than its calculated mass (54 kDa) , which is important for proper interpretation of results.

How do I troubleshoot inconsistent results with ETV4 antibodies across different experimental systems?

Inconsistent results with ETV4 antibodies can stem from multiple factors that require systematic troubleshooting:

Biological variability sources:

• Alternative splicing: Expression of different isoforms varies across cell types

• Post-translational modifications: Phosphorylation patterns may differ between systems

• Expression level variations: ETV4 is highly expressed in some cancers but barely detectable in others

• Nuclear localization efficiency: Can vary by cell type or condition

Technical considerations:

• Sample preparation: Nuclear extraction may be necessary for consistent detection

• Extraction buffers: Include appropriate protease/phosphatase inhibitors

• Antibody batch variability: Especially relevant for polyclonal antibodies

• Detection systems: Sensitivity requirements may vary by application

Systematic troubleshooting approach:

• Validate with positive and negative controls (BxPC-3 cells as positive; MCF7 cells as negative)

• Test multiple antibodies targeting different epitopes

• Compare with mRNA expression data to confirm biological variability

• Optimize protocols specifically for each experimental system

Documentation for reproducibility:

• Record complete antibody information (supplier, catalog number, lot, dilution)

• Document exact protocol conditions (incubation times, temperatures, buffers)

• Report specific bands observed and their molecular weights

• Note any deviations from expected results

A study of ETV4 splice variants found significant variation in alternative transcript expression between cell lines, with alternative transcripts accounting for 23% of total ETV4 mRNA in PC3 cells but up to 74% in 22RV1 cells . This biological variability highlights the importance of comprehensive validation across experimental systems.

How are ETV4 antibodies being used in emerging cancer biomarker research?

ETV4 antibodies are increasingly utilized in biomarker development, with several promising research directions:

Prognostic biomarker applications:

• Tissue microarray studies show correlation between ETV4 expression and patient survival in multiple cancers

• Integration with other biomarkers may improve prognostic accuracy

• ETV4 expression correlates with tumor heterogeneity markers and stemness indices

Predictive biomarker potential:

• ETV4 expression has been linked to drug sensitivity profiles, suggesting utility in therapy selection

• Combined analysis with immune checkpoint markers may predict immunotherapy response

• ETV4 status may inform selection of targeted therapies

Emerging multiplex approaches:

• Cytometric bead arrays with ETV4 antibody pairs enable liquid biopsy applications

• Multiplex immunofluorescence panels incorporate ETV4 with other cancer markers

• Mass cytometry with metal-conjugated antibodies allows high-dimensional analysis

Technical innovations:

• Recombinant antibody technology provides superior batch consistency

• Ready-to-conjugate formats facilitate custom multiplex panel development

• Validation in multiple assay contexts enhances translational potential

A recent pan-cancer analysis demonstrated that ETV4 expression is associated with poor prognosis across multiple cancer types and correlates with molecular features including mismatch repair genes, DNA methyltransferases, and immune cell infiltration . These findings suggest that ETV4 antibodies will play an increasingly important role in comprehensive cancer biomarker panels.

What are the current challenges in developing phospho-specific ETV4 antibodies?

Development of phospho-specific ETV4 antibodies faces several technical challenges despite their potential value:

Key phosphorylation sites:

• ETV4 is regulated by phosphorylation, particularly through MAPK pathway signaling

• Multiple phosphorylation sites exist, with varying functional significance

• Identification of the most biologically relevant sites requires extensive validation

Technical hurdles:

• Phosphopeptide immunogen design: Must ensure specificity and accessibility

• Cross-reactivity: Related ETS family members share sequence homology

• Confirmation of specificity: Requires phosphatase treatment and mutant controls

• Preservation of phosphorylation during sample preparation: Critical for accurate detection

Validation requirements:

• Phosphomimetic and phospho-null mutants as controls

• Mass spectrometry confirmation of modification sites

• In vitro kinase assays to establish modification conditions

• Signal loss after phosphatase treatment

Applications awaiting phospho-specific antibodies:

• Pathway activation monitoring in response to therapies

• Correlation of specific phosphorylation events with functional outcomes

• Dynamic regulation studies during cancer progression

• Identification of patients likely to respond to MAPK pathway inhibitors

Current research relies on mobility shift detection (54 kDa → 61-65 kDa) to infer phosphorylation status , but site-specific phospho-antibodies would enable more precise analysis of ETV4 regulation. The development of such antibodies represents an important opportunity for advancing ETV4 research.

How can ETV4 antibodies contribute to understanding its role in therapy resistance?

ETV4 has been implicated in therapy resistance across multiple cancer types, and antibody-based approaches offer valuable insights:

Monitoring ETV4 in treatment response:

• Serial biopsies analyzed by IHC can track changes in ETV4 expression during treatment

• Correlation with treatment response may identify ETV4 as a resistance marker

• Western blot analysis of cell line models before and after drug exposure reveals adaptation mechanisms

Mechanistic studies:

• Co-immunoprecipitation to identify interaction partners in resistant vs. sensitive cells

• ChIP-seq to map altered genomic binding in resistant states

• Protein localization changes in response to therapy can be tracked by immunofluorescence

Overcoming resistance strategies:

• ETV4 knockdown/inhibition in combination with primary therapies

• Analysis of downstream targets as alternative intervention points

• Identification of synthetic lethal interactions in ETV4-high contexts

Clinical implications:

• IHC-based patient stratification for clinical trials

• Monitoring circulating tumor cells for ETV4 expression as a liquid biopsy approach

• Development of ETV4-targeted therapeutics for resistant cancers

Research has shown that ETV4 may promote cancer drug resistance through multiple mechanisms, including regulation of immune checkpoint genes and promotion of cancer stemness properties . Studies linking ETV4 to drug sensitivity profiles suggest that monitoring its expression and activity may inform treatment selection and combination strategies to overcome resistance.