EHF Antibody

What is EHF and why is it significant in research?

EHF (Ets homologous factor) is a transcriptional activator that regulates epithelial cell differentiation and proliferation. It functions as a repressor for specific ETS/AP-1-responsive genes and modulates nuclear response to mitogen-activated protein kinase signaling cascades. EHF binds to DNA sequences containing the consensus nucleotide core sequence GGAA and is involved in regulating TNFRSF10B/DR5 expression through Ets-binding sequences. Notably, EHF may contribute to development and carcinogenesis by acting as a tumor suppressor gene or anti-oncogene . Its critical roles in epithelial function make it an important research target across multiple disease contexts, including cancer and respiratory diseases .

What types of EHF antibodies are available for research, and what are their validated applications?

There are multiple types of EHF antibodies available for research with different host species and validated applications:

Researchers should select the appropriate antibody based on their specific experimental needs. For Western blot applications, both mouse and rabbit polyclonal antibodies have been validated, while for immunohistochemistry and ChIP applications, rabbit polyclonal antibodies have demonstrated efficacy in research contexts .

What are the recommended dilutions for EHF antibodies in common applications?

Based on validated research protocols, the following dilutions are recommended for optimal results with EHF antibodies:

It's important to note that optimal dilutions may be sample-dependent. Researchers are advised to titrate the antibody in their specific testing systems to obtain optimal results .

What are the appropriate positive controls for EHF antibody validation?

For proper validation of EHF antibodies, researchers should consider the following positive controls:

• For Western blot: LNCaP cells and A431 cells have been confirmed to express detectable levels of EHF protein

• For immunohistochemistry: Human ovary cancer tissue has been validated as a positive control

• For transfection studies: Comparing EHF 293T cell line transfected lysate with non-transfected controls can verify antibody specificity

Always include appropriate negative controls in your experimental design to ensure the specificity of the antibody binding.

How can EHF antibodies be effectively used in ChIP-seq experiments to identify genome-wide binding sites?

ChIP-seq with EHF antibodies has been successfully employed to generate genome-wide binding signatures for EHF in primary human bronchial epithelial (HBE) cells. The methodology involves:

• Crosslinking protein-DNA complexes in intact cells

• Sonicating chromatin to appropriate fragment sizes

• Immunoprecipitating with a specific EHF antibody (previously validated for ChIP applications)

• Sequencing the immunoprecipitated DNA

In a key study, this approach identified 11,326 peaks with an irreproducible discovery rate (IDR) < 0.05, using sonicated input DNA as background control. The normalized tag counts at each called peak showed significant correlation between biological replicates (r = 0.29, p < 0.0001) .

To validate direct or indirect targets, researchers can complement ChIP-seq with EHF depletion experiments in the same cell type, followed by RT-qPCR to measure expression changes in potential target genes. This approach has revealed that EHF regulates the expression of several important transcription factors, including HOPX, KLF5, RARB, and SPDEF .

What is the significance of EHF as a biomarker in cancer research, and how should immunohistochemical evaluation be performed?

EHF has emerged as a potential prognostic biomarker for prostate cancer metastasis formation, independent of Gleason scoring systems. Research has demonstrated that primary prostate lesions with ≥40% EHF-positive cells exhibit significantly higher risk of developing metastasis within five years of initial diagnosis .

For immunohistochemical evaluation of EHF in cancer tissues:

• Perform antigen retrieval with EDTA citrate pH 7.8 for 30 minutes at 95°C

• Incubate sections with rabbit polyclonal anti-EHF antibody (1:200; ab272671, ABCAM) for 1 hour at room temperature

• Use PBS/Tween20 pH 7.6 for washing steps

• Reveal reactions with HRP-DAB Detection Kit

• Evaluate immunoreaction as percentage of EHF positive cells on the whole tissue section

In a cohort study of 152 prostate biopsies, immunohistochemical analysis revealed significant differences in EHF expression between different prostate cancer types:

Logistic regression analysis established that samples with ≥40% EHF-positive cells had approximately 40-fold increased risk of developing metastasis compared to those with ≤30% positive cells .

How can EHF expression be experimentally manipulated to study its function in cellular models?

Researchers can manipulate EHF expression through several experimental approaches:

• Overexpression studies:

  • Clone the full-length open reading frame (ORF) of human EHF into a mammalian expression vector (e.g., pcDNA3.1/myc-His(-) with or without tags)

  • Transfect the construct into appropriate cell lines using standard transfection methods

  • Validate expression by Western blot and/or qRT-PCR before conducting functional assays

• Knockdown experiments:

  • Design and validate siRNAs targeting EHF (e.g., si-EHF-979)

  • Transiently transfect siRNAs into cell lines expressing EHF

  • Confirm knockdown efficiency at both mRNA and protein levels

  • Observe phenotypic changes (such as effects on xenograft tumor growth within 15 days post-transfection)

• ChIP assays to identify direct targets:

  • Transfect cells with tagged EHF expression constructs (e.g., Myc-tagged)

  • Perform ChIP using anti-tag antibodies (e.g., anti-Myc)

  • Analyze binding to specific promoters by qRT-PCR

  • This approach has successfully identified direct binding of EHF to the HER2 promoter, with three different promoter fragments (P1: −604/−484; P2: −274/−155; P3: −147/−37) showing 8.14-fold enrichment on average in EHF-transfected cells compared to control

What are the key considerations for quantifying EHF expression at the mRNA level?

For accurate quantification of EHF expression at the mRNA level:

• Extract total RNA using appropriate reagents (e.g., Trizol)

• Synthesize cDNA with 500 ng total RNA using reverse transcription kits

• Perform qRT-PCR using SYBR-based methods with EHF-specific primers

• Normalize expression to appropriate reference genes (e.g., 18S rRNA)

• Run each sample in triplicate to ensure statistical validity

When comparing EHF expression across different experimental conditions or tissue types, ensure consistent RNA quality and quantity. Additionally, validate qRT-PCR findings with protein expression analysis when possible, as post-transcriptional regulation may affect the correlation between mRNA and protein levels.

How should researchers address potential non-specific binding when using EHF antibodies?

Non-specific binding is a common challenge when working with antibodies. To address this issue with EHF antibodies:

• Always include appropriate negative controls (samples known not to express EHF or isotype controls)

• Use the recommended antibody dilutions and incubation conditions

• Optimize blocking conditions to reduce background staining

• For Western blots, consider using lower antibody concentrations and including detergents in washing buffers

• For immunohistochemistry, test different antigen retrieval methods if background is high

If multiple bands appear in Western blot experiments, validate which band represents the true EHF protein by comparing with positive control samples and checking against the expected molecular weight (approximately 35 kDa) . Additionally, consider testing the antibody in EHF-knockout or knockdown samples to confirm specificity.

How can researchers reconcile conflicting data about EHF function across different tissue types?

EHF appears to have context-dependent functions across different tissue types and disease states. For example, it may act as a tumor suppressor in some contexts and potentially contribute to cancer progression in others . To address conflicting data:

• Consider tissue-specific cofactors: EHF may interact with different partners in different cell types

• Examine isoform expression: Alternative splicing may generate different EHF variants with distinct functions

• Investigate the activation state of relevant signaling pathways: The effect of EHF may depend on the cellular context and active signaling networks

• Perform comprehensive ChIP-seq and RNA-seq analyses: To identify tissue-specific binding sites and gene regulation patterns

• Use multiple experimental models: Validate findings across different cell lines and in vivo models

When contradictory results emerge, design experiments that directly compare EHF function across multiple contexts within the same experimental framework to minimize technical variables.

What are the potential pitfalls when interpreting EHF immunohistochemistry results in clinical samples?

When interpreting EHF immunohistochemistry in clinical samples, researchers should be aware of several potential pitfalls:

For prostate cancer specifically, research has shown that a threshold of 40% EHF-positive cells may distinguish patients at high risk for metastasis, but this threshold should be validated in independent cohorts before clinical application .

How is EHF involved in transcriptional networks regulating epithelial function?

EHF functions within complex transcriptional networks that regulate epithelial cell differentiation and function. ChIP-seq studies have revealed that EHF binds to numerous sites throughout the genome, regulating the expression of other transcription factors critical for epithelial biology .

Key findings about EHF in transcriptional networks include:

• EHF depletion significantly affects the expression of other transcription factors including HOPX, KLF5, RARB (increased expression), and SPDEF (decreased expression)

• EHF may indirectly influence CEBPG and FOXA1 expression, though with substantial variation between primary cultures

• EHF binds directly to the HER2 promoter, providing a mechanism for its role in certain cancers

These findings suggest that EHF sits at a crucial node in epithelial transcriptional networks, with its expression level potentially serving as a switch that determines cell fate and function in diverse epithelial tissues.

What are the emerging applications of EHF antibodies in single-cell analysis techniques?

While the provided search results don't specifically address single-cell applications, the growing importance of single-cell techniques suggests several potential applications for EHF antibodies:

• Single-cell protein profiling: Using highly specific EHF antibodies in mass cytometry (CyTOF) or microfluidic-based single-cell Western blotting

• Spatial transcriptomics combined with immunohistochemistry: Correlating EHF protein expression with transcriptional profiles at single-cell resolution within tissue contexts

• ChIP-seq at single-cell level: Though technically challenging, emerging protocols for single-cell ChIP-seq could reveal cell-to-cell variation in EHF binding patterns

• Proximity ligation assays: Identifying EHF protein interaction partners at single-cell resolution

These applications would require validation of antibody specificity and sensitivity in the context of single-cell techniques, which typically work with much smaller amounts of starting material than conventional bulk approaches.

How might advances in antibody technology enhance EHF research in the coming years?

The field of antibody technology continues to evolve rapidly, offering several potential advancements that could benefit EHF research:

• Development of monoclonal antibodies: While current research heavily utilizes polyclonal antibodies , the development of highly specific monoclonal antibodies against EHF could improve reproducibility and reduce batch-to-batch variation

• Recombinant antibody fragments: Smaller antibody formats like Fab fragments or nanobodies could provide superior tissue penetration for imaging applications

• Antibody-based proximity labeling: Techniques like BioID or APEX2 fused to anti-EHF antibodies could help identify transient protein interactions in living cells

• Multiplexed immunofluorescence: Advanced multiplex platforms could enable simultaneous visualization of EHF with multiple other markers to better understand its role in complex cellular networks

• Engineered antibodies with enhanced properties: Modifications to improve stability, reduce non-specific binding, or add functional capabilities (such as photoswitchable fluorophores)

These technological advances, combined with the growing understanding of EHF biology, should enable more sophisticated investigations into the role of this important transcription factor in health and disease.

What key questions about EHF function remain to be addressed in future research?

Despite significant progress in understanding EHF, several important questions remain:

• How does EHF expression and function change during development and in response to environmental stressors?

• What is the complete set of direct genomic targets of EHF across different cell types, and how does this binding profile change in disease states?

• What post-translational modifications regulate EHF activity, and how do these modifications affect its function?

• How does EHF cooperate with or antagonize other transcription factors to fine-tune gene expression programs?

• Can EHF expression or activity be therapeutically manipulated to treat diseases like cancer or inflammatory conditions?