ire1 Antibody

What is IRE1α and why is it important in cellular research?

IRE1α is a key protein that functions as a stress sensor in the unfolded protein response (UPR), a cellular mechanism activated when misfolded or unfolded proteins accumulate in the endoplasmic reticulum (ER). This protein plays a crucial role in maintaining cellular homeostasis and preventing apoptosis under stress conditions . IRE1α is localized in the ER lumen and contains a transmembrane domain spanning the ER membrane, along with a cytosolic Ser/Thr kinase domain that becomes activated during ER stress . Its importance in research stems from its central role in cellular signaling pathways related to stress responses, with significant implications for cancer biology and aging-related conditions .

What detection methods can be used with IRE1α antibodies?

IRE1α antibodies, such as the mouse monoclonal IRE1α Antibody (B-12), can be utilized across multiple detection methods including:

• Western blotting (WB) for protein expression analysis

• Immunoprecipitation (IP) for protein-protein interaction studies

• Immunofluorescence (IF) for subcellular localization visualization

• Enzyme-linked immunosorbent assay (ELISA) for quantitative detection

For optimal results, researchers should select antibody conjugations appropriate for their specific application, as IRE1α antibodies are available in non-conjugated forms and various conjugated forms including agarose, horseradish peroxidase (HRP), phycoerythrin (PE), fluorescein isothiocyanate (FITC), and Alexa Fluor® conjugates .

How does IRE1α function in the unfolded protein response pathway?

Under normal conditions, IRE1α exists predominantly as dimers in unstressed cells . When ER stress occurs, IRE1α undergoes a conformational change that leads to oligomerization and trans-phosphorylation, enhancing its kinase activity . This activation triggers a signaling cascade resulting in the transcription of UPR-targeted genes essential for protein folding and restoration of ER function . Additionally, IRE1α participates in the phosphorylation of Jun N-Terminal Kinase (JNK), activating the cellular MAP kinase pathway . Recent research has demonstrated that IRE1α oligomers are smaller than previously thought and disassemble back into dimers after the stress response is resolved .

What genetic engineering approaches can be used to study IRE1α dynamics in living cells?

Advanced researchers can employ CRISPR/Cas9-based gene editing to insert tags like HaloTag into the IRE1 genomic locus, allowing for real-time tracking of endogenous IRE1 molecules . When implementing this approach:

• Insert a C-terminal HaloTag into IRE1's genomic locus using CRISPR/Cas9

• Select clones based on comparable expression to unedited cells

• Verify absence of wild-type IRE1 protein lacking the tag

• Confirm intact UPR activation in response to ER stress

• Validate expression levels through comparison with endogenous IRE1

This approach enables quantitative assessment of IRE1 oligomerization states and dynamics in response to stress conditions while maintaining near-endogenous expression levels, avoiding artifacts associated with overexpression systems .

How can researchers differentiate between IRE1α dimers and oligomers experimentally?

Distinguishing between IRE1α dimers and oligomers requires specialized techniques:

• Single-molecule tracking in living cells using HaloTag-labeled IRE1α

• Trajectory analysis to determine molecular movement patterns

• Comparison of diffusion coefficients between stress and non-stress conditions

• Application of mutational analysis targeting specific domains

Research by Belyy et al. demonstrated that IRE1α proteins generally exist as dimers in unstressed cells and temporarily assemble into oligomers during ER stress before disassembling back into dimers . This insight was gained through a novel microscopy approach that could count tagged IRE1 molecules in living cells, revealing that oligomerization is a transient state rather than a permanent feature of stress response .

What are the key domains involved in IRE1α oligomerization and how can they be targeted experimentally?

The IRE1α oligomerization process involves specific protein domains that can be experimentally manipulated:

Researchers can use these mutations to dissect the relationship between oligomerization and signaling. The K121Y mutation disrupts the IF1L interface and prevents dimer formation, while the WLLI 359-362-GSGS mutation disrupts the IF2L interface, preventing stress-induced oligomerization but maintaining dimeric structure . These targeted approaches allow researchers to separate the roles of dimerization and oligomerization in IRE1α signaling.

How does IRE1α influence antigen presentation in dendritic cells?

Recent research has revealed a novel mechanism by which IRE1α regulates immune responses through dendritic cells (DCs):

• Antigen-derived hydrophobic peptides can directly engage ER-resident IRE1α, mimicking unfolded proteins

• This engagement activates IRE1α even in the absence of conventional ER stress

• Activated IRE1α depletes MHC-I heavy-chain mRNAs through regulated IRE1α-dependent decay (RIDD)

• This depletion curtails antigen cross-presentation to CD8+ T cells

This mechanism represents an unexpected connection between IRE1α and adaptive immunity, suggesting that IRE1α activation in DCs may serve as a regulatory checkpoint for immune responses. Researchers studying this pathway should consider examining antigen properties, particularly hydrophobicity, when investigating cross-presentation efficiency .

What experimental approaches can be used to study IRE1α in cancer immunotherapy contexts?

Researchers investigating IRE1α as a potential target for cancer immunotherapy can employ these methodological approaches:

• Use IRE1α-specific kinase inhibitors in tumor-bearing mouse models

• Analyze MHC-I expression levels on tumor-infiltrating DCs

• Monitor CD8+ T cell recruitment and activation within the tumor microenvironment

• Assess synergistic effects when combining IRE1α inhibition with immune checkpoint blockade (e.g., anti-PD-L1 therapy)

Studies have demonstrated that blocking IRE1α function in tumor-bearing mice upregulates MHC-I levels on DCs, enhances tumor recruitment and activation of CD8+ T cells, and cooperates with anti–PD-L1 immune-checkpoint disruption to cause tumor regression . These findings highlight the potential for combining IRE1α inhibition with existing immunotherapies to improve clinical outcomes.

How can researchers validate the specificity of IRE1α antibodies?

To ensure experimental rigor when working with IRE1α antibodies, researchers should implement these validation steps:

• Include appropriate positive controls (e.g., cells known to express IRE1α)

• Incorporate negative controls (e.g., IRE1α knockout cells generated via CRISPR/Cas9)

• Confirm antibody specificity through multiple detection methods (WB, IP, IF)

• Verify target band molecular weight matches the expected size of IRE1α with consideration for any post-translational modifications

• When possible, use multiple antibodies targeting different epitopes of IRE1α

Comparing expression patterns between different cell types and under various stress conditions can further validate antibody specificity while providing biologically relevant insights.

What are common pitfalls when studying IRE1α oligomerization and how can they be addressed?

Several experimental challenges can confound IRE1α oligomerization studies:

• Expression level artifacts: High overexpression can lead to artificial clustering that doesn't represent physiological conditions. Use endogenously tagged IRE1α or controlled expression systems to maintain near-native levels .

• Fluorescent protein fusion effects: Some fluorescent tags may influence oligomerization behavior. Control experiments with different tags or tag-free approaches should be considered .

• Cell type specificity: IRE1α behavior may vary across different cell types. Research has shown that certain mechanisms of IRE1α engagement might be specific to particular cells, such as bone marrow-derived dendritic cells .

• Temporal dynamics confusion: The transient nature of IRE1α oligomerization requires appropriate time-course experiments to capture the full dynamics of assembly and disassembly .

Researchers should carefully control protein expression levels, use multiple complementary techniques to validate observations, and consider the specific cellular context when interpreting results.

How can IRE1α antibodies be utilized to study aging-related conditions?

IRE1α signaling has been implicated in aging processes and age-related diseases. Researchers can leverage IRE1α antibodies to:

• Map age-dependent changes in IRE1α expression and activation across tissues

• Investigate connections between chronic ER stress, IRE1α signaling, and age-related cellular dysfunction

• Explore potential interventions targeting the IRE1α pathway to mitigate age-related pathologies

• Examine the intersection of IRE1α activity with other aging-related pathways such as inflammaging and proteostasis decline

Given IRE1α's role in cellular stress responses and its implications in aging biology, antibody-based approaches can provide valuable insights into how ER stress responses evolve throughout lifespan and contribute to age-related diseases.

What novel microscopy techniques can be combined with IRE1α antibodies for advanced research?

Cutting-edge microscopy approaches can enhance IRE1α research:

• Single-molecule tracking with HaloTag-labeled IRE1α allows precise quantification of oligomerization states in living cells

• Super-resolution microscopy techniques (PALM, STORM, STED) can resolve IRE1α clusters below the diffraction limit

• Förster resonance energy transfer (FRET) microscopy can detect IRE1α protein-protein interactions

• Correlative light and electron microscopy (CLEM) can connect IRE1α dynamics with ultrastructural changes in the ER membrane

The microscopy approach developed by Belyy et al. to count IRE1 molecules in living cells represents a significant advancement that could be adapted to study other proteins involved in transmembrane signaling .