ero1a Antibody

What is ERO1A and why is it an important research target?

ERO1A is a glycoprotein that mediates oxidative formation of protein disulfide bonds in the endoplasmic reticulum. It has gained significant research interest due to its critical roles in multiple cellular processes including hypoxic response, cancer progression, and immune regulation. In humans, ERO1A is a 468 amino acid protein with a mass of 54.4 kDa that localizes primarily to the ER and Golgi apparatus, although it can also be secreted . ERO1A has been implicated in tumor formation, integrin glycosylation, membrane transport in hypoxic conditions, and modulation of the immune microenvironment . Its overexpression correlates with poor prognosis in several cancers, making it an important target for both basic research and potential therapeutic development .

What are the key considerations when selecting an ERO1A antibody for research?

When selecting an ERO1A antibody, researchers should consider several factors: (1) Target specificity - ensure the antibody specifically recognizes ERO1A without cross-reactivity with its paralog ERO1β; (2) Species reactivity - verify compatibility with your experimental model, as ERO1A orthologs exist in multiple species including mouse, rat, bovine, and others; (3) Application suitability - confirm the antibody has been validated for your specific application (Western blot, IHC, ELISA, etc.); (4) Epitope location - consider whether the antibody targets regions that may be affected by post-translational modifications like N-glycosylation and phosphorylation; and (5) Clone type - determine whether a monoclonal or polyclonal antibody better suits your research question . For studies examining ERO1A in cancer microenvironments, antibodies validated under hypoxic conditions would be particularly valuable .

How can I validate the specificity of my ERO1A antibody?

Proper validation is essential to ensure experimental rigor. A comprehensive validation approach includes: (1) Positive and negative control testing - using tissues/cells known to express high levels of ERO1A (such as cerebral cortex or cervix) versus those with minimal expression; (2) Knockout validation - testing the antibody on ERO1A knockout samples created via CRISPR/Cas9 targeting exon 1, which has been successfully used to eliminate ERO1A expression while preserving ERO1β ; (3) Peptide competition assays - pre-incubating the antibody with purified ERO1A protein to confirm signal reduction; (4) Multiple detection methods - confirming results across different techniques (Western blot, IHC, IF); and (5) Subcellular localization confirmation - verifying the expected ER/Golgi localization pattern. This multi-layered approach minimizes the risk of misinterpretation due to antibody non-specificity .

How can ERO1A antibodies be used to study tumor immunology?

ERO1A antibodies serve as powerful tools for investigating the complex relationship between ERO1A expression and anti-tumor immunity. Researchers can employ these antibodies for: (1) Characterizing the tumor microenvironment through multiplex immunofluorescence to correlate ERO1A expression with immune cell infiltration patterns; (2) Assessing ERO1A's association with immunosuppressive signaling by examining co-localization with PD-L1 and other immune checkpoint molecules; (3) Quantifying ERO1A expression in patient samples to predict response to immunotherapy, as high ERO1A expression correlates with resistance to PD-1 blockade; (4) Monitoring changes in ERO1A expression following therapeutic interventions; and (5) Evaluating ERO1A's relationship with immunogenic cell death pathways induced by endoplasmic reticulum stress . Recent studies have demonstrated that ERO1A mediates an immune-suppressive tumor microenvironment by impairing the balance between IRE1α and PERK signaling activities, making this protein a promising target for enhancing immunotherapy efficacy .

What role does ERO1A play in hypoxic tumor microenvironments, and how can antibodies help study this?

ERO1A is hypoxia-inducible and plays critical roles in tumor cell adaptation to hypoxic conditions. ERO1A antibodies enable researchers to: (1) Track upregulation of ERO1A in response to hypoxia through time-course immunoblotting or immunostaining; (2) Investigate ERO1A's role in integrin glycosylation and membrane transport processes that support cancer cell survival under hypoxia; (3) Correlate ERO1A expression with hypoxic markers (HIF-1α, CA9) in tumor sections; (4) Examine ERO1A's impact on the secretome of hypoxic tumor cells through immunoprecipitation followed by mass spectrometry; and (5) Assess how ERO1A inhibition affects hypoxia-driven biological pathways . Studies have shown that inhibition of ERO1A significantly downregulates hypoxic pathways (by approximately 12.5-fold compared to untreated controls) while upregulating antigen presentation pathways, suggesting a key mechanistic link between ERO1A and hypoxia-driven immune evasion .

What are the best methods for detecting ERO1A in different experimental systems?

Optimal detection methods vary based on experimental objectives:

For most research applications, Western blotting remains the gold standard for ERO1A detection, with immunohistochemistry being particularly valuable for translational research using patient samples .

How should ERO1A knockout models be designed and validated using antibodies?

Creating and validating ERO1A knockout models requires careful design and rigorous validation:

• Design Strategy: Target exon 1 of ERO1A using CRISPR/Cas9 to ensure complete protein elimination while preserving ERO1β expression. This approach has been successfully employed in colorectal cancer cell lines (HCT116) .

• Validation Steps:

  • Confirm genomic modification through sequencing of the targeted region

  • Verify complete protein elimination via Western blot with antibodies targeting different ERO1A epitopes

  • Assess ERO1β expression to confirm specificity (should remain unchanged)

  • Evaluate phenotypic changes under both normoxic and hypoxic conditions (ERO1A KO cells show marked differences only in hypoxic environments)

  • Confirm functional consequences through appropriate assays (e.g., disulfide bond formation)

• Functional Analysis:

  • Compare proliferation rates between WT and KO cells under normoxia vs. hypoxia

  • Assess morphological changes (KO cells maintain cell-cell integrity under hypoxia)

  • Examine impact on tumor formation using xenograft models (ERO1A KO cells show diminished tumorigenicity)

  • Investigate alterations in immune infiltration in tumor models

Properly validated knockout models combined with specific antibody detection provide powerful tools for understanding ERO1A's biological functions .

What protocols are recommended for studying ERO1A's role in immunogenic cell death?

Investigating ERO1A's involvement in immunogenic cell death (ICD) requires specialized protocols:

• ER Stress Induction Methods:

  • Hypoxia-induced stress (1% O₂)

  • Metabolic stress (glucose deprivation)

  • Pharmacological induction (tunicamycin treatment)

• Cell Death Assessment:

  • Measure LDH release to quantify cell death (ERO1A KO tumors show increased LDH release compared to WT)

  • Assess cell viability under ER-stressed conditions

  • Monitor morphological changes via microscopy

• ICD Marker Detection:

  • Surface calreticulin exposure (flow cytometry)

  • HMGB1 release (ELISA)

  • ATP secretion (luminescence assay)

• Immune Response Evaluation:

  • Analyze tumor-infiltrating lymphocytes (TILs) via immunofluorescence staining

  • Quantify CD4⁺, CD8⁺, and NK cells in the tumor microenvironment

  • Measure cytokine production (IFN-γ, TNF-α, IL-1β, IL-6) using Luminex-based multiplexing

• Mechanistic Analysis:

  • Examine UPR pathway activation (IRE1α and PERK signaling) via Western blot

  • Assess PD-L1 expression patterns (focal expression pattern observed in ERO1A KO tumors)

  • Evaluate IFN-γ-responsive gene signatures

These methodologies enable comprehensive characterization of how ERO1A modulates ER stress responses and subsequent immunogenic cell death, which is critical for its role in anti-tumor immunity .

How can ERO1A antibodies be used to investigate its interactions with calcium signaling molecules?

ERO1A has been identified as a critical regulator of calcium signaling through direct interactions with key calcium modulators. Researchers can employ antibodies to:

• Protein-Protein Interaction Studies:

  • Perform co-immunoprecipitation experiments to confirm direct interactions between ERO1A and calcium signaling molecules STIM1 and SERCA2

  • Use proximity ligation assays to visualize these interactions in situ

  • Conduct pulldown assays with mutant proteins (STIM1-Cys49/56Ser, SERCA2-Cys875/887Ser) to validate specific interaction sites

• Functional Analysis:

  • Examine ERO1A's thiol oxidase activity on STIM1 (Cys49-Cys56) and SERCA2 (Cys875-Cys887) disulfide bonds

  • Monitor calcium store content and cytosolic calcium levels during cell activation in the presence/absence of ERO1A

  • Assess the impact of ERO1A inhibitors on calcium mobilization

• Structural Studies:

  • Use antibodies for immunoelectron microscopy to precisely localize ERO1A within the dense tubular system

  • Map the domains of ERO1A involved in STIM1/SERCA2 interactions

• Therapeutic Applications:

  • Test small-molecule ERO1A inhibitors vs. blocking antibodies for effects on calcium signaling

  • Evaluate downstream functional consequences in thrombosis and ischemic stroke models

These approaches have revealed that ERO1A functions as a thiol oxidase for calcium signaling molecules, enhancing cytosolic calcium levels and promoting cellular activation - insights with significant implications for thrombotic disease therapies .

What approaches can be used to study the effects of ERO1A inhibition on dendritic cell function?

Investigating ERO1A inhibition effects on dendritic cells requires multifaceted approaches:

• Tumor-Immune Cell Co-culture Systems:

  • Establish PDAC spheroid models for studying monocyte infiltration and differentiation

  • Use ERO1A antibodies to confirm inhibitor efficacy and track expression changes

  • Combine ERO1A inhibition with IDO1 inhibition to assess synergistic effects

• Secretome Analysis:

  • Perform LC-MS/MS analysis of tumor spheroid secretomes following ERO1A inhibition

  • Quantify changes in immunomodulatory factors (IL-8, lymphocyte cytosolic protein 1, transgelin-2)

  • Use pathway analysis to identify altered biological functions

• Transcription Factor Profiling:

  • Evaluate changes in myeloid-associated transcription factors upon ERO1A inhibition

  • Monitor downregulation of BACH1 and ARID3A (associated with non-inflammatory M2 macrophages)

  • Track upregulation of SP1 and KLF7 (involved in myeloid activity and macrophage activation)

• Functional Dendritic Cell Assays:

  • Assess antigen presentation capacity following ERO1A inhibition

  • Measure T cell activation in DC co-culture systems

  • Evaluate DC maturation markers and cytokine production

Research has demonstrated that combined inhibition of ERO1A and IDO1 significantly upregulates antigen presentation biological pathways (by approximately 14.59%) while downregulating hypoxic pathways, creating conditions favorable for dendritic cell infiltration and function .

How can researchers study ERO1A's role in regulating PD-L1 expression and immunotherapy response?

Investigating the relationship between ERO1A and immunotherapy response requires sophisticated methodologies:

• Expression Correlation Studies:

  • Use multiplexed immunohistochemistry to assess co-expression of ERO1A, PD-L1, and immune markers in patient samples

  • Analyze large datasets (e.g., TCGA) to identify correlations between ERO1A and immune checkpoint molecules

  • Track ERO1A expression in samples from patients undergoing immunotherapy

• Mechanistic Investigations:

  • Examine PD-L1 expression patterns in ERO1A knockout vs. wild-type tumors (focal expression patterns observed in KO tumors)

  • Assess IFN-γ production by tumor-infiltrating lymphocytes as a potential mediator

  • Investigate the UPR pathway's role in linking ERO1A activity to PD-L1 regulation

• In Vivo Response Models:

  • Evaluate anti-PD-1 therapy efficacy in ERO1A-knockout vs. wild-type tumor models

  • Characterize immune infiltrate composition using flow cytometry

  • Monitor cytokine production (IFN-γ, TNF-α, IL-1β, IL-6) in the tumor microenvironment

• Clinical Correlation:

  • Assess ERO1A expression in human lung cancer samples to predict recurrence risk following neoadjuvant immunotherapy

  • Develop predictive models combining ERO1A status with other biomarkers

Research has shown that ERO1A ablation substantially enhances anti-tumor T cell immunity and improves response to PD-1 blockade in therapeutic models. This is associated with higher tumor-infiltrating lymphocyte abundance and increased PD-L1 expression (likely due to IFN-γ production by activated T cells), suggesting ERO1A as a valuable predictive biomarker and potential therapeutic target .

What are common challenges in detecting ERO1A and how can they be addressed?

Researchers frequently encounter several challenges when working with ERO1A antibodies:

Implementing these troubleshooting approaches will improve the reliability and reproducibility of ERO1A detection across experimental platforms .

How should researchers interpret ERO1A expression data in the context of hypoxia and ER stress?

Proper interpretation of ERO1A expression data requires careful consideration of experimental context:

• Baseline vs. Induced Expression:

  • ERO1A is upregulated under hypoxia and ER stress conditions

  • Normoxic cultures may show minimal expression, leading to false negative results

  • Always include appropriate hypoxic/stressed positive controls

• Temporal Dynamics:

  • ERO1A induction follows specific kinetics after stress onset

  • Conduct time-course experiments to capture expression peaks

  • Consider relationships between acute vs. chronic stress responses

• Cell Type Variations:

  • Expression patterns differ substantially between cell types

  • Cancer cells often show constitutive expression compared to normal counterparts

  • Interpret results within the specific cellular context

• Subcellular Localization:

  • ERO1A can redistribute between ER, Golgi, and secreted fractions

  • Changes in localization may occur without alterations in total expression

  • Use fractionation and imaging to assess compartment-specific changes

• Relationship to Functional Outcomes:

  • Connect expression data to functional readouts (proliferation, survival under stress)

  • Consider ERO1A's relationship with downstream effectors (STIM1, SERCA2)

  • Validate biological significance through gain/loss of function experiments

Research has demonstrated that while ERO1A knockout cells appear morphologically normal under standard conditions, they exhibit dramatically reduced proliferation and altered cell-cell contacts specifically under hypoxic conditions, highlighting the context-dependent nature of ERO1A function .

What emerging therapeutic approaches target ERO1A, and how can antibodies support their development?

Several promising therapeutic strategies targeting ERO1A are under investigation:

• Small Molecule Inhibitors:

  • More effective than blocking antibodies in reducing arterial thrombosis and ischemic stroke in mouse models

  • Can attenuate infarct volume following focal brain ischemia

  • Antibodies are essential for validating target engagement and specificity

• Combination Therapies:

  • ERO1A and IDO1 dual inhibition shows synergistic effects on antigen presentation pathways

  • Antibodies help monitor pathway modulation and resistance mechanisms

  • Support patient stratification based on ERO1A expression levels

• Immunotherapy Enhancement:

  • ERO1A ablation substantially improves response to PD-1 blockade

  • Antibodies facilitate biomarker development and response prediction

  • Enable monitoring of treatment-induced changes in tumor microenvironment

• Targeting Specific Protein-Protein Interactions:

  • Disrupting ERO1A interactions with STIM1 and SERCA2 may offer more selective approaches

  • Structure-guided drug design benefits from antibody-based structural studies

• Delivery Systems:

  • Tumor-targeted delivery of ERO1A inhibitors

  • Antibody-drug conjugates or bispecific antibodies could enhance specificity

Recent studies have demonstrated that ERO1A inhibition can significantly reshape the tumor microenvironment, making it more conducive to immune infiltration and effective immunotherapy - insights that would not have been possible without the development of specific antibodies to track these changes .

How might ERO1A research contribute to understanding and treating ischemic diseases?

ERO1A's newly discovered role in thrombotic conditions opens important research avenues:

• Platelet Activation Mechanisms:

  • ERO1A is exclusively localized in the platelet dense tubular system

  • Promotes calcium mobilization, platelet activation, and aggregation

  • Antibodies enable precise localization and mechanistic studies

• Therapeutic Target Validation:

  • ERO1A deficiency reduces platelet thrombus formation without affecting bleeding times

  • Small-molecule inhibitors show promise in reducing thrombotic events

  • Antibodies essential for confirming target specificity and expression patterns

• Calcium Signaling Pathways:

  • ERO1A directly interacts with and modifies STIM1 and SERCA2

  • Forms specific disulfide bonds (Cys49-Cys56 in STIM1, Cys875-Cys887 in SERCA2)

  • Antibody-based proximity assays help map these interactions

• Translational Models:

  • Megakaryocyte-specific or global deletion models show similar phenotypes

  • Intravital microscopy with fluorescently-labeled antibodies enables real-time monitoring

  • Patient stratification based on ERO1A expression could identify high-risk individuals

• Multi-Organ Protection Strategies:

  • ERO1A inhibition reduces infarct volume following focal brain ischemia

  • Potential applications beyond stroke to other thrombotic conditions

  • Antibodies support tissue-specific expression analysis

The discovery that ERO1A functions as a thiol oxidase for calcium signaling molecules represents a significant advancement in understanding thrombotic disease mechanisms and offers new therapeutic approaches with potentially favorable bleeding risk profiles .