EHD1 Antibody

What is EHD1 and why is it important in cellular biology?

EHD1 is a membrane-shaping protein involved in critical cellular processes including endocytic vesicle trafficking and ciliogenesis. It functions primarily through cooperation with various binding partners, including members of the Rab family of small GTPases, which regulate endocytic trafficking . EHD1 is widely expressed across human tissues, with highest expression in brain, respiratory system, kidney, and male reproductive system, particularly the testes . Its importance lies in:

• Regulating post-endocytic recycling of cell surface receptors back to the cell surface

• Facilitating pre-activation transport of newly-synthesized receptor tyrosine kinases (RTKs) from the Golgi to plasma membrane

• Contributing to membrane fission events in the endocytic pathway

• Playing essential roles in kidney proximal tubule function and auditory system development

In research contexts, EHD1 antibodies are essential tools for studying these fundamental cellular processes.

How do mutations in EHD1 correlate with disease states?

Several significant disease associations with EHD1 mutations have been identified:

• Tubular Proteinuria and Hearing Loss: A homozygous missense mutation (c.1192C>T; p.R398W) in EHD1 has been identified in patients with low-molecular-weight proteinuria (0.7–2.1 g/d) and high-frequency hearing loss . This mutation results in:

  • Decreased proximal tubular reabsorption of filtered proteins

  • Impaired membrane fission events in tubular recycling endosomes

  • Formation of abnormal elongated tubular structures in cells

• Cancer Progression: EHD1 overexpression is linked to Ewing sarcoma progression :

  • High EHD1 mRNA expression correlates with shorter patient survival

  • 88.6% of evaluated EWS patient samples show high EHD1 protein expression

  • EHD1 expression is significantly higher in metastases compared to primary tumors

Understanding these pathological associations makes EHD1 antibodies valuable tools for both basic research and translational medicine.

What are the critical considerations when selecting an EHD1 antibody?

When selecting an EHD1 antibody for research applications, consider:

• Epitope specificity: Choose antibodies that distinguish EHD1 from other EHD family members (EHD2, EHD3, EHD4), as these proteins share significant sequence homology .

• Application compatibility: Verify validation data for your specific application (Western blot, IHC, IF, IP).

• Species reactivity: Confirm cross-reactivity with your experimental model organism. The search results show successful use of EHD1 antibodies in both human and mouse tissues .

• Mutation detection: For studies involving EHD1 variants (e.g., R398W), ensure the antibody recognizes the mutant form. The search results demonstrate that antibodies can detect differentially localized mutant EHD1 .

• Validation controls: Look for antibodies validated using knockout controls. The literature shows confirmation of antibody specificity using Ehd1−/− mice .

How can researchers definitively validate EHD1 antibody specificity?

Rigorous validation is essential for reliable EHD1 research:

• Genetic knockout controls: The gold standard approach, as demonstrated in the search results where Ehd1 knockout mice (Ehd1−/−) were used to confirm antibody specificity .

• Knockdown verification: Compare staining between control and EHD1-knockdown cells (using siRNA or shRNA approaches) .

• Multiple antibody concordance: Use antibodies targeting different EHD1 epitopes and confirm consistent results.

• Recombinant protein controls: Test reactivity against purified EHD1 versus other EHD family members.

• Species-specific validation: For cross-species studies, verify antibody reactivity in each species separately.

The search results demonstrate this approach where researchers confirmed "Ehd1 knockout and antibody specificity by the presence or absence of Ehd1 staining in wildtype and Ehd1−/− mice, respectively" .

What are the optimal conditions for immunohistochemical detection of EHD1?

Based on published protocols in the literature:

• Tissue fixation:

  • Paraformaldehyde (4%) fixation is recommended for optimal epitope preservation

  • Avoid prolonged fixation which may mask epitopes

• Antigen retrieval:

  • Heat-induced epitope retrieval with citrate buffer (pH 6.0)

  • Critical for detecting membrane-associated proteins like EHD1

• Blocking and antibody conditions:

  • 5-10% normal serum with 0.1-0.3% Triton X-100

  • Primary antibody dilutions typically 1:100-1:500, incubate overnight at 4°C

  • Secondary antibody incubation for 1-2 hours at room temperature

• Visualization strategies:

  • For co-localization studies, fluorescent secondary antibodies are preferred

  • For quantitative assessment, enzymatic detection (HRP/DAB) may be used

• Essential controls:

  • Positive control: Tissues with known high EHD1 expression (kidney, testes)

  • Negative control: EHD1 knockout tissue or secondary-only control

  • Multiple antibody validation: Compare staining patterns with different antibodies

These approaches were successfully used to demonstrate EHD1 localization in human kidney, where it was found "predominantly in the subapical compartment of proximal tubular epithelial cells" .

How can EHD1 antibodies be optimized for co-localization studies with trafficking markers?

Co-localization studies are essential for understanding EHD1's trafficking functions:

Research has successfully employed these approaches to show that in mouse kidney, "Ehd1 partially colocalized with the endocytic tracer β2-microglobulin and the apical receptor proteins megalin and cubilin" .

What methods are most effective for studying mutant EHD1 protein trafficking defects?

Studies of EHD1 mutations require specialized approaches:

• Expression systems:

  • Transfection of wild-type versus mutant EHD1 (e.g., R398W)

  • CRISPR/Cas9 knock-in of point mutations

  • Lentiviral expression systems for stable cell lines

• Visualization strategies:

  • Live-cell imaging of fluorescently tagged EHD1 variants

  • Fixed-cell confocal microscopy with antibody detection

  • Electron microscopy for ultrastructural analysis

• Functional trafficking assays:

  • Receptor internalization and recycling kinetics

  • Fluorescent cargo trafficking (e.g., transferrin, β2-microglobulin)

  • Golgi-to-plasma membrane transport rates

• Data analysis approaches:

  • Quantification of abnormal structures (size, number, distribution)

  • Colocalization with trafficking markers

  • Live-cell trafficking dynamics (speed, directionality, residence time)

These methods revealed that cells expressing mutated EHD1 (R398W) "showed elongated tubular structures, presumably tubular recycling endosomes, indicating an impairment of membrane fission events" .

How are EHD1 antibodies used to investigate cancer progression mechanisms?

EHD1 antibodies have provided critical insights into cancer biology:

• Expression analysis in patient tissues:

  • Immunohistochemistry using EHD1 antibodies on tissue microarrays revealed that 88.6% of 307 evaluable Ewing sarcoma samples showed high EHD1 expression

  • Higher EHD1 expression was detected in metastases compared to primary tumors, suggesting a role in disease progression

• Mechanistic investigations:

  • EHD1 antibodies helped identify IGF-1R as a trafficking target of EHD1 regulation in Ewing sarcoma

  • Immunofluorescence studies demonstrated EHD1's role in endocytic recycling and Golgi-to-plasma membrane transport of IGF-1R

• Therapeutic target assessment:

  • EHD1 knockdown/knockout studies revealed reduced cell proliferation, migration, and invasion in vitro

  • Reduced tumorigenesis and metastasis in vivo after EHD1 depletion

  • Rescue experiments with mouse Ehd1 confirmed EHD1's specific role in these processes

• Molecular pathway analysis:

  • Immunoblotting with EHD1 and RTK antibodies established their regulatory relationship

  • RNA-seq analysis showed reduced cell cycle regulatory gene expression after EHD1 knockdown

These approaches established that "EHD1-dependent endocytic recycling and pre-activation Golgi to the plasma membrane traffic of IGF-1R are essential for its oncogenic role" .

What experimental designs best demonstrate EHD1's role in receptor trafficking?

Comprehensive experimental designs include:

• Surface biotinylation assays:

  • Biotinylate cell surface proteins

  • Allow internalization and recycling for various time points

  • Use EHD1 antibodies to immunoprecipitate EHD1 complexes

  • Analyze biotinylated receptors associated with EHD1

• Receptor trafficking kinetics:

  • Compare wild-type and EHD1-depleted cells

  • Measure receptor internalization, recycling, and degradation rates

  • Quantify receptor half-life and steady-state distribution

• Golgi-to-plasma membrane transport:

  • Temperature-sensitive VSVG-GFP reporter system

  • Track newly synthesized receptor transport in presence/absence of functional EHD1

  • Quantify arrival rates at the cell surface

• Multi-color live imaging:

  • Co-express fluorescently tagged EHD1 and receptors

  • Use spinning disk confocal or TIRF microscopy

  • Track co-migration of vesicles in real-time

Research using these approaches has demonstrated that EHD1 regulates "the post-endocytic recycling of a variety of cell surface receptors back to the cell surface" and "pre-activation transport of newly-synthesized RTKs from the Golgi to the plasma membrane" .

How should researchers interpret conflicting results between EHD1 knockout and mutation models?

Discrepancies between knockout and mutation models require systematic analysis:

• Mechanistic differences:

  • Complete absence (knockout) versus dysfunctional protein (mutation)

  • Potential dominant-negative effects of mutations

  • Compensatory upregulation of other EHD family members in knockout models

• Experimental approach for resolution:

  • Compare protein expression levels of all EHD family members (EHD1-4)

  • Assess localization patterns using antibodies against each family member

  • Perform rescue experiments with wild-type versus mutant EHD1

• Function-specific analysis:

  • Compare specific cellular functions (e.g., receptor recycling, ciliogenesis)

  • Evaluate structure-function relationships through domain mutation studies

  • Conduct interaction partner analysis in both models

• Developmental considerations:

  • Account for potential developmental compensation in knockout models

  • Use conditional or inducible knockout systems to minimize adaptation

The search results provide an example where researchers used both Ehd1 knockout mice (Ehd1−/−) and R398W knockin mice (Ehd1R398W/R398W) to comprehensively characterize EHD1 function in kidney, finding similar but not identical phenotypes .

What factors influence EHD1 antibody detection of post-translational modifications?

Post-translational modifications (PTMs) can affect antibody detection:

• Common EHD1 modifications:

  • Phosphorylation: Can alter protein conformation and epitope accessibility

  • Ubiquitination: May signal for degradation

  • SUMOylation: Can affect protein localization and function

• Antibody selection considerations:

  • Modification-specific antibodies: Recognize only modified forms

  • Modification-sensitive antibodies: Epitope masked by modifications

  • Modification-insensitive antibodies: Detect both modified and unmodified forms

• Sample preparation factors:

  • Phosphatase inhibitors: Preserve phosphorylation status

  • Denaturing conditions: May expose hidden epitopes

  • Native conditions: Maintain tertiary structure-dependent epitopes

• Validation approaches:

  • Treatment with modifying/demodifying enzymes

  • Mutation of modification sites

  • Mass spectrometry verification of modification state

The search results note that the EHD1 R398W mutant migrates differently on SDS-PAGE than wild-type EHD1, appearing as a "distinctly slower-migrating" band , which could reflect altered protein folding or post-translational modifications.

How can researchers address conflicting localization patterns observed with different EHD1 antibodies?

When different antibodies produce inconsistent localization patterns:

• Systematic evaluation protocol:

  • Compare antibody epitopes: Different regions may be accessible in different cellular compartments

  • Test multiple fixation methods: Cross-linking vs. precipitating fixatives

  • Vary permeabilization conditions: Different detergents expose different epitopes

  • Control for specificity using knockout tissues/cells

• Technical resolution approaches:

  • Super-resolution microscopy for precise localization

  • Immuno-electron microscopy for ultrastructural localization

  • Subcellular fractionation with Western blotting

  • Co-localization with definitive compartment markers

• Complementary non-antibody methods:

  • Fluorescent protein tagging (N- vs. C-terminal tags)

  • CRISPR knock-in of epitope tags at endogenous loci

  • Proximity labeling approaches (BioID or APEX)

• Data integration framework:

  • Triangulate results from multiple methods

  • Weight evidence based on control quality

  • Consider biological context and known functions

The search results demonstrate effective validation by confirming "Ehd1 knockout and antibody specificity by the presence or absence of Ehd1 staining in wildtype and Ehd1−/− mice, respectively" , providing a gold standard control approach.