IARS2 Antibody

What is IARS2 and what cellular function does it serve?

IARS2 (Isoleucyl-tRNA Synthetase 2) is a nuclear-encoded enzyme that functions within mitochondria as an aminoacyl-tRNA synthetase. It specifically catalyzes the attachment of isoleucine to its cognate tRNA (tRNA-Ile), serving a critical role in mitochondrial protein translation . This enzyme belongs to the highly conserved family of aminoacyl-tRNA synthetases that ensure proper amino acid incorporation during protein synthesis. The protein has a predicted molecular weight of approximately 114 kDa and contains functional domains including an anticodon binding region in its C-terminus . IARS2 is essential for proper mitochondrial function and protein synthesis, with mutations in this gene being linked to several distinct clinical presentations including syndromic disorders affecting multiple organ systems .

How does IARS2 expression vary across different cell types?

IARS2 is expressed across diverse human cell types, though with variable expression levels. Based on laboratory findings using western blot analysis, IARS2 protein has been detected in multiple cell lines including HeLa (human cervical adenocarcinoma cells), HEK-293T (human embryonic kidney cells), and Jurkat (human T-cell leukemia) cell lines . In cancer research, IARS2 has been found to be expressed in multiple melanoma cell lines including A375, MUM-2B, and C918, as demonstrated through both RT-qPCR and protein analysis techniques . The ubiquitous expression pattern of IARS2 reflects its fundamental role in mitochondrial translation, though its relative abundance may differ across tissues. This variability should be considered when designing experiments that target IARS2 in specific cell types, particularly when selecting appropriate positive controls for antibody validation.

What criteria should be considered when selecting an IARS2 antibody for research applications?

When selecting an IARS2 antibody for research applications, researchers should evaluate several critical parameters:

• Epitope specificity: Select antibodies targeting well-conserved regions of IARS2. For example, antibodies targeting the C-terminal region (such as those recognizing epitopes within amino acids 950 to C-terminus) have demonstrated reliable detection in multiple applications .

• Validated applications: Verify that the antibody has been validated for your specific application (Western blot, immunoprecipitation, immunohistochemistry, etc.). For instance, some IARS2 antibodies have been specifically validated for Western blot (WB) and immunoprecipitation (IP) applications in human samples .

• Species reactivity: Confirm cross-reactivity with your species of interest. While many IARS2 antibodies are optimized for human samples, cross-reactivity with other species may be predicted based on sequence homology but should be experimentally validated .

• Clonality: Consider whether a monoclonal or polyclonal antibody is more suitable for your application. Polyclonal antibodies like the rabbit polyclonal ab242116 may provide robust signal detection across multiple epitopes .

• Published validation data: Review available data including Western blot images, predicted band sizes (approximately 114 kDa for IARS2), and positive control recommendations (such as HeLa, HEK-293T, or Jurkat cell lysates) .

Thorough evaluation of these criteria will help ensure reliable and reproducible results in IARS2-focused research.

What are the recommended validation procedures for confirming IARS2 antibody specificity?

To rigorously validate IARS2 antibody specificity, researchers should implement a multi-step validation protocol:

• Western blot analysis with multiple controls:

  • Positive controls: Use cell lines with confirmed IARS2 expression (HeLa, HEK-293T, Jurkat cell lysates at approximately 15 μg loading) .

  • Predicted band size verification: Confirm detection at the expected molecular weight (~114 kDa) .

  • Exposure time optimization: Start with moderate exposure (e.g., 30 seconds) and adjust as needed .

• RNA interference validation:

  • Implement shRNA or siRNA knockdown of IARS2 as demonstrated in melanoma studies where lentiviral-mediated IARS2 knockdown significantly reduced both mRNA and protein expression .

  • Compare antibody signal between knockdown and control samples to confirm signal reduction correlates with decreased IARS2 expression.

• Immunoprecipitation validation:

  • Perform IP using the IARS2 antibody followed by Western blot detection to confirm specific pulldown of the target protein .

  • Consider using 1 mg of protein per IP reaction with approximately 20% of immunoprecipitated material loaded for detection .

• Dilution optimization:

  • Test a range of antibody dilutions to determine optimal signal-to-noise ratio (e.g., 1/1000 dilution has been effective for Western blot applications) .

• Detection method variation:

  • Compare results using different detection systems (e.g., ECL technique has proven effective for IARS2 visualization) .

Documentation of these validation steps is essential for ensuring reproducibility and reliability in subsequent experiments involving IARS2 antibodies.

What are the optimal conditions for Western blot detection of IARS2?

For optimal Western blot detection of IARS2, researchers should adhere to the following protocol based on validated experimental conditions:

• Sample preparation:

  • Prepare whole cell lysates using NETN lysis buffer, which has been validated for IARS2 extraction .

  • Use established cell lines as positive controls: HeLa, HEK-293T, or Jurkat cells.

  • Load approximately 15 μg of total protein per lane for standard detection .

• Electrophoresis and transfer parameters:

  • Use gel concentration appropriate for detecting the 114 kDa IARS2 protein (8-10% acrylamide gels are typically suitable).

  • Ensure complete transfer of high molecular weight proteins using appropriate transfer conditions (longer transfer times or specialized buffers for large proteins).

• Blocking and antibody incubation:

  • Primary antibody: Use a 1/1000 dilution of anti-IARS2 antibody (e.g., ab242116) .

  • Incubation conditions: Typically overnight at 4°C or according to manufacturer's recommendations.

  • Secondary antibody: Select based on primary antibody host species (anti-rabbit for rabbit polyclonal antibodies).

• Detection optimization:

  • The ECL technique has been successfully employed for IARS2 visualization .

  • Start with a moderate exposure time of approximately 30 seconds, then adjust as needed based on signal intensity .

  • Expect a band at approximately 114 kDa, which is the predicted molecular weight of IARS2 .

These conditions have been experimentally validated to produce clear and specific detection of IARS2 protein in human cell lines and can serve as a starting point for researchers developing their own Western blot protocols.

How can IARS2 antibodies be applied in studying IARS2-related disease mechanisms?

IARS2 antibodies serve as valuable tools for investigating disease mechanisms related to IARS2 dysfunction through multiple experimental approaches:

• Expression analysis in disease models:

  • Comparative analysis of IARS2 protein levels between normal and pathological tissues can reveal alterations associated with disease states.

  • In cancer research, IARS2 protein expression has been examined in melanoma cell lines where it appears to play a role in cell proliferation and survival .

• Functional studies in patient-derived samples:

  • IARS2 antibodies can be used to assess protein expression in patient samples harboring IARS2 mutations, such as those identified in CAGSSS syndrome (cataracts, growth hormone deficiency, sensory neuropathy, sensorineural hearing loss, and skeletal dysplasia) .

  • Researchers studying biallelic IARS2 mutations in sideroblastic anemia can utilize these antibodies to evaluate protein expression and potential functional consequences of variants like c.2025dup (p.Asp676*) and c.986T>C (p.Leu329Pro) .

• Mechanistic investigation using knockdown/knockout models:

  • IARS2 antibodies have been employed to confirm successful knockdown in RNA interference experiments, as demonstrated in studies where shRNA-mediated IARS2 knockdown significantly reduced protein levels in A375 melanoma cells .

  • These knockdown models revealed that IARS2 depletion inhibits proliferation, induces apoptosis (increasing from 2.62±0.35% to 8.77±1.32%), and disrupts cell cycle progression in melanoma cells .

• Co-immunoprecipitation for protein interaction studies:

  • IARS2 antibodies can facilitate the investigation of protein interaction networks through co-immunoprecipitation experiments, helping elucidate how IARS2 mutations might disrupt critical protein-protein interactions .

• Mitochondrial function assessment:

  • Given IARS2's role in mitochondrial translation, antibodies can be used in conjunction with analyses of mitochondrial respiratory chain complex formation and activity in patient samples .

These applications demonstrate how IARS2 antibodies can substantially contribute to understanding the molecular basis of IARS2-related pathologies and potentially identify therapeutic targets.

How should researchers address non-specific binding when using IARS2 antibodies?

Non-specific binding is a common challenge when working with antibodies that can compromise data interpretation. When using IARS2 antibodies, researchers should implement the following systematic troubleshooting approaches:

• Optimization of blocking conditions:

  • Experiment with different blocking agents (BSA, non-fat dry milk, commercial blocking buffers) to identify optimal conditions that minimize background while preserving specific IARS2 signal.

  • Consider extending blocking time (1-2 hours at room temperature or overnight at 4°C) to reduce non-specific interactions.

• Antibody dilution adjustment:

  • Titrate the primary antibody concentration beyond the standard 1/1000 dilution used for Western blot applications .

  • Prepare a dilution series (e.g., 1/500, 1/1000, 1/2000, 1/5000) to determine the optimal concentration that maximizes signal-to-noise ratio.

• Stringent washing protocols:

  • Implement additional and longer washing steps with buffers containing increased detergent concentrations (0.1-0.3% Tween-20 or Triton X-100).

  • Consider using TBS rather than PBS for phospho-specific applications or antibodies sensitive to phosphate buffers.

• Validation with IARS2 knockdown controls:

  • Generate IARS2 knockdown samples as negative controls, similar to the approach used in melanoma studies where shRNA-mediated IARS2 depletion significantly reduced specific signal .

  • Any bands that remain unchanged in knockdown samples likely represent non-specific binding targets.

• Alternative antibody evaluation:

  • When persistent non-specific binding occurs, test alternative IARS2 antibodies that recognize different epitopes.

  • Compare polyclonal antibodies (which may exhibit more non-specific binding but stronger signals) with monoclonal options (potentially higher specificity).

By methodically applying these approaches, researchers can substantially reduce non-specific binding issues when working with IARS2 antibodies, leading to more reliable and interpretable experimental results.

What considerations are important when studying post-translational modifications of IARS2?

Investigating post-translational modifications (PTMs) of IARS2 requires specialized methodological considerations:

• Phosphorylation analysis:

  • Preserve phosphorylation states by including phosphatase inhibitors (sodium fluoride, sodium orthovanadate, β-glycerophosphate) in lysis buffers.

  • Consider phospho-enrichment techniques (phospho-protein/peptide enrichment columns, immunoprecipitation with phospho-specific antibodies) prior to IARS2 detection.

  • Validate potential phosphorylation sites through phosphatase treatment controls that should eliminate phospho-specific signals.

• Ubiquitination and SUMOylation detection:

  • Include deubiquitinating enzyme inhibitors (N-ethylmaleimide, iodoacetamide) in lysis buffers.

  • Consider using specialized systems that stabilize ubiquitin conjugates by inhibiting the proteasome (MG132) prior to cell lysis.

  • Perform sequential immunoprecipitation: first for IARS2, then immunoblot for ubiquitin/SUMO, or vice versa.

• Acetylation assessment:

  • Include deacetylase inhibitors (trichostatin A, nicotinamide) in lysis buffers.

  • Consider MS/MS-based approaches to map specific acetylation sites on immunoprecipitated IARS2.

• Mitochondrial import processing:

  • Since IARS2 is a nuclear-encoded mitochondrial protein, investigate potential processing of its mitochondrial targeting sequence through size comparison of cytosolic precursor versus mature mitochondrial forms.

  • Consider subcellular fractionation to separate cytosolic and mitochondrial populations before immunoblotting.

• Oxidative modification considerations:

  • Given IARS2's mitochondrial localization, it may be susceptible to oxidative modifications. Include reducing agents (DTT, β-mercaptoethanol) to prevent artificial oxidation during sample preparation.

  • Consider comparative analysis under oxidative stress conditions to evaluate potential redox-sensitive modifications.

These specialized approaches enable researchers to comprehensively characterize the post-translational landscape of IARS2, which may reveal regulatory mechanisms relevant to both normal function and disease states associated with IARS2 mutations.

What cellular phenotypes are associated with IARS2 knockdown or mutation?

IARS2 disruption produces distinct cellular phenotypes that have been experimentally characterized:

• Proliferation defects:

  • Knockdown of IARS2 in A375 melanoma cells significantly impairs cell growth as demonstrated through cell counting assays over a 5-day period .

  • MTT proliferation assays confirm growth inhibition in IARS2-depleted cells compared to control cells .

  • Colony formation capacity is substantially reduced following IARS2 knockdown (quantitative data shows statistically significant reduction, p<0.05) .

• Apoptotic induction:

  • Flow cytometry analysis using Annexin V-APC/7-AAD staining reveals increased apoptosis in IARS2-knockdown melanoma cells (8.77±1.32%) compared to control cells (2.62±0.35%) .

  • Elevated caspase 3/7 activity has been observed in IARS2-depleted cells, confirming apoptotic pathway activation .

• Cell cycle disruption:

  • IARS2 knockdown leads to cell cycle arrest with increased proportion of cells in G0/G1 phase and decreased proportion in S phase .

  • This cell cycle redistribution suggests IARS2 plays a role in regulating G1/S transition.

• Mitochondrial dysfunction in patient cells:

  • Patient-derived cells harboring pathogenic IARS2 variants have been assessed for mitochondrial function .

  • While some patients show normal respiratory chain enzyme activities despite IARS2 mutations , the functional impact may manifest in tissue-specific or stress-induced contexts.

• Disease-specific cellular phenotypes:

  • In sideroblastic anemia associated with IARS2 mutations, cellular phenotypes include dysfunctional iron utilization in erythroid precursors .

  • Cells from patients with the CAGSSS syndrome may exhibit tissue-specific manifestations of IARS2 dysfunction in neuronal, sensory, and skeletal tissues .

These experimentally characterized phenotypes provide valuable insights into the cellular consequences of IARS2 disruption and establish cellular models that can be utilized to study disease mechanisms and potential therapeutic interventions.

How does the subcellular localization of IARS2 influence experimental design with IARS2 antibodies?

The mitochondrial localization of IARS2 introduces important considerations for experimental design:

• Subcellular fractionation requirements:

  • When studying IARS2, researchers should consider implementing mitochondrial isolation protocols to enrich for the relevant subcellular compartment.

  • Pure mitochondrial fractions can be prepared using differential centrifugation followed by density gradient separation to maximize IARS2 detection sensitivity.

  • Validation of fraction purity should include immunoblotting for compartment-specific markers (e.g., VDAC or COX IV for mitochondria, tubulin for cytosol).

• Immunofluorescence considerations:

  • When performing immunofluorescence studies, co-localization with established mitochondrial markers (MitoTracker dyes, TOM20, COX IV) is essential for confirming the specificity of IARS2 antibody staining.

  • Fixation and permeabilization methods should be optimized for mitochondrial proteins (2-4% paraformaldehyde with 0.1-0.3% Triton X-100 is often suitable).

  • High-resolution microscopy techniques (confocal, super-resolution) may be necessary to distinguish mitochondrial IARS2 from potential cytosolic populations.

• Import pathway analysis:

  • As a nuclear-encoded mitochondrial protein, IARS2 requires translocation into mitochondria, which may result in processing of the N-terminal mitochondrial targeting sequence.

  • Antibodies targeting different epitopes (N-terminal vs. C-terminal) may yield different results depending on whether they recognize the precursor form, the mature form, or both.

• Lysis buffer optimization:

  • Standard RIPA or NETN buffers may be insufficient for complete extraction of mitochondrial proteins.

  • Consider stronger lysis conditions that effectively solubilize mitochondrial membranes (buffers containing 1-2% Triton X-100, digitonin, or specialized mitochondrial extraction reagents).

• Proximity-based protein interaction studies:

  • When investigating IARS2 protein interactions, techniques that preserve the mitochondrial spatial context (e.g., proximity ligation assay, BioID, or APEX labeling) may provide more physiologically relevant results than standard co-immunoprecipitation approaches.

By accounting for these mitochondrial-specific considerations, researchers can develop more effective experimental protocols for studying IARS2 biology and avoid potential pitfalls associated with its subcellular compartmentalization.

How can IARS2 antibodies be utilized to investigate the role of IARS2 in cancer progression?

Recent studies suggest IARS2 may function as a cancer-promoting gene, presenting several strategic approaches for antibody-based investigation:

• Expression profiling across cancer types:

  • IARS2 antibodies can be employed for immunohistochemical analysis of tissue microarrays spanning multiple cancer types to establish expression patterns.

  • Evidence already indicates that IARS2 expression is detectable in melanoma cell lines (A375, MUM-2B, C918) , suggesting potential relevance in skin cancer.

  • Researchers should develop quantitative scoring systems for IARS2 staining intensity and subcellular localization patterns to correlate with clinical parameters.

• Mechanistic investigation of proliferation and apoptosis:

  • Building on findings that IARS2 knockdown inhibits melanoma cell proliferation and promotes apoptosis , researchers can investigate the underlying molecular mechanisms.

  • IARS2 antibodies enable protein analysis in experimental systems where proliferation pathways (MAPK, PI3K/AKT) and apoptotic markers (cleaved caspase-3, PARP cleavage) are assessed following manipulation of IARS2 levels.

  • The observed cell cycle arrest in G0/G1 phase following IARS2 knockdown warrants investigation of cell cycle regulators (cyclins, CDKs) in relation to IARS2 function.

• Metabolic reprogramming assessment:

  • Given IARS2's mitochondrial function, antibodies can be used to study its potential role in cancer-associated metabolic reprogramming.

  • Researchers should correlate IARS2 expression with metabolic pathway proteins and consider how IARS2 alterations might influence the Warburg effect or glutamine addiction in cancer cells.

• Mutation and variant analysis:

  • The finding that approximately 59% of colorectal cancer patients harbor mutations in the 5' upstream region of IARS2 suggests regulatory disruption may be important.

  • Antibodies can help determine whether these mutations alter protein expression levels or post-translational modifications.

• Therapeutic response prediction:

  • IARS2 antibodies could be used to evaluate whether expression levels correlate with sensitivity to specific cancer therapies, particularly those targeting mitochondrial function or protein synthesis.

  • Given the apoptotic effects of IARS2 knockdown, researchers might investigate whether IARS2 expression predicts response to apoptosis-inducing therapies.

These research directions could significantly advance our understanding of IARS2's role in cancer biology and potentially identify new therapeutic targets or biomarkers.

What advancements in antibody technology might enhance future IARS2 research?

Emerging antibody technologies offer significant potential to advance IARS2 research beyond current capabilities:

• Single-domain antibodies and nanobodies:

  • The development of camelid-derived single-domain antibodies could enable better access to conformational epitopes within IARS2's structure.

  • These smaller antibody formats may provide superior penetration into mitochondrial compartments where IARS2 functions, potentially improving imaging resolution and subcellular localization studies.

• Conformation-specific antibodies:

  • Engineering antibodies that specifically recognize distinct conformational states of IARS2 could reveal functional changes associated with substrate binding or catalytic activity.

  • Such tools would be particularly valuable for understanding how disease-causing mutations (like those identified in CAGSSS syndrome or sideroblastic anemia) might alter protein conformation.

• Degradation-inducing antibody conjugates:

  • Proteolysis-targeting chimeras (PROTACs) or antibody-based equivalents could selectively target IARS2 for degradation, offering more complete and rapid protein elimination than traditional RNAi approaches.

  • These technologies would provide temporal control over IARS2 depletion, enabling precise studies of acute versus chronic loss effects.

• Intrabodies for live-cell imaging:

  • Genetically encoded antibody fragments expressed intracellularly could track IARS2 dynamics in living cells.

  • When combined with fluorescent protein tags, these constructs could monitor IARS2 localization changes during stress responses or mitochondrial dysfunction.

• Combinatorial epitope detection systems:

  • Multiplexed antibody-based detection systems could simultaneously monitor IARS2 alongside interaction partners or downstream effectors.

  • Technologies like multiplexed ion beam imaging (MIBI) or co-detection by indexing (CODEX) could reveal spatial relationships between IARS2 and other proteins at subcellular resolution.

• Antibody engineering for reduced cross-reactivity:

  • Machine learning approaches to antibody design could generate IARS2-targeting antibodies with minimal cross-reactivity to related aminoacyl-tRNA synthetases.

  • This would be particularly valuable given the sequence similarity within this enzyme family and potential for misleading results due to off-target binding.

These technological advances represent promising directions that could significantly enhance the specificity, versatility, and information content derived from antibody-based IARS2 research.

How do results from IARS2 antibody studies correlate with genetic and clinical findings?

Integration of antibody-based protein studies with genetic and clinical data provides comprehensive insights into IARS2-related pathologies:

This integrated approach enables researchers to:

• Establish genotype-phenotype correlations at the protein level

• Identify tissue-specific vulnerability factors that explain the selective manifestation of IARS2 defects

• Develop targeted therapeutic approaches based on mechanistic understanding of disease pathways

• Design personalized monitoring strategies for patients with specific IARS2 variants

What experimental controls are essential when comparing data from different IARS2 antibodies?

When comparing results from different IARS2 antibodies, researchers must implement rigorous control measures to ensure valid data interpretation:

• Epitope mapping verification:

  • Determine the precise epitopes recognized by each antibody, particularly important when comparing antibodies targeting different regions (N-terminal vs. C-terminal domains).

  • Antibodies recognizing different epitopes may yield discrepant results if certain domains are masked by protein interactions or subject to post-translational modifications.

• Validation across multiple detection methods:

  • Cross-validate findings using orthogonal techniques (e.g., if Western blot shows discrepancies between antibodies, verify with immunoprecipitation or mass spectrometry).

  • Include positive controls with confirmed IARS2 expression (HeLa, HEK-293T, Jurkat cells) for each antibody tested.

• IARS2 knockdown/knockout controls:

  • Generate IARS2-depleted samples through RNAi or CRISPR approaches as demonstrated in melanoma studies .

  • All antibodies should show corresponding signal reduction in these samples, with any persistent signals indicating potential non-specific binding.

• Recombinant protein standards:

  • Use purified recombinant IARS2 protein at defined concentrations as standard curves to assess relative affinity and sensitivity of different antibodies.

  • This approach enables quantitative comparison of detection efficiency across antibodies.

• Application-specific validation:

  • An antibody performing well in Western blot may not be suitable for immunoprecipitation or immunohistochemistry.

  • Validate each antibody specifically for the intended application rather than assuming cross-application reliability.

• Cross-laboratory standardization:

  • When comparing results between research groups, document detailed protocols including:

    • Antibody source, catalog number, lot number, and dilution

    • Sample preparation methods (lysis buffers, fixatives)

    • Blocking conditions and incubation parameters

    • Detection systems and image acquisition settings

By implementing these controlled comparison approaches, researchers can more confidently integrate findings from multiple antibody-based studies and minimize technical variability that might otherwise be misinterpreted as biological significance.

What are the current limitations in IARS2 antibody research and how might they be addressed?

Current IARS2 antibody research faces several significant limitations that require strategic approaches to overcome:

• Limited epitope diversity:

  • Current commercial antibodies often target similar epitopes, particularly in the C-terminal region (aa 950 to C-terminus) .

  • Recommendation: Develop antibodies targeting diverse functional domains including the aminoacylation domain, editing domain, and anticodon-binding domain to enable comprehensive protein characterization.

• Insufficient validation in disease models:

  • While IARS2 has been implicated in multiple conditions (CAGSSS syndrome, sideroblastic anemia, cancer) , antibody validation in these specific disease contexts remains limited.

  • Recommendation: Validate antibodies using patient-derived materials and disease models, with particular attention to potential conformational changes in mutant proteins.

• Inconsistent subcellular fractionation approaches:

  • Given IARS2's mitochondrial localization, variations in mitochondrial isolation protocols may affect detection efficiency.

  • Recommendation: Establish standardized mitochondrial enrichment protocols optimized for IARS2 detection and document fractionation efficiency using established markers.

• Cross-reactivity with related aminoacyl-tRNA synthetases:

  • The aminoacyl-tRNA synthetase family contains highly conserved domains that may lead to antibody cross-reactivity.

  • Recommendation: Implement rigorous specificity testing using recombinant proteins representing related family members and validate in cells with selective knockdown of IARS2 versus related synthetases.

• Limited temporal and spatial resolution:

  • Current approaches provide static snapshots rather than dynamic information about IARS2 behavior during cellular processes.

  • Recommendation: Develop live-cell imaging approaches using fluorescently tagged antibody fragments or integrate antibody-based detection with emerging spatial transcriptomics technologies.

• Incomplete correlation with functional outcomes:

  • Detection of IARS2 protein levels often lacks parallel assessment of enzymatic activity.

  • Recommendation: Combine antibody-based detection with functional assays measuring aminoacylation activity to establish structure-function relationships, particularly in the context of disease-associated variants.

Addressing these limitations will substantially advance the utility of IARS2 antibodies as tools for basic research and clinical applications, ultimately enhancing our understanding of IARS2-related pathologies.

What emerging research questions about IARS2 could be addressed using advanced antibody-based approaches?

Advanced antibody-based methodologies could address several compelling research questions about IARS2 biology and pathology:

• Mitochondrial stress response dynamics:

  • Research question: How does IARS2 expression, localization, and modification change during mitochondrial stress conditions?

  • Approach: Time-course immunofluorescence studies using conformation-specific antibodies combined with mitochondrial stress markers following exposure to mitochondrial toxins or in patient-derived cells.

• Tissue-specific vulnerability mechanisms:

  • Research question: Why do IARS2 mutations affect specific tissues (eyes, inner ear, peripheral nerves) despite ubiquitous expression?

  • Approach: Spatial proteomics using tissue microarrays with quantitative IARS2 antibody staining correlated with tissue-specific interaction partners and compensatory mechanisms.

• Cancer progression and therapeutic resistance:

  • Research question: Does IARS2 contribute to metabolic adaptation during cancer progression and therapy resistance?

  • Approach: Proximity-labeling approaches using IARS2 antibodies to identify context-specific interaction networks in treatment-naive versus resistant cancer cells, building on observations of IARS2's role in melanoma proliferation .

• Translational regulation during cellular stress:

  • Research question: How does IARS2 function change during integrated stress response activation?

  • Approach: Ribosome profiling combined with IARS2 immunoprecipitation to identify translational targets specifically affected by IARS2 dysfunction.

• Therapeutic targeting potential:

  • Research question: Can IARS2 inhibition selectively target cancer cells with mitochondrial vulnerabilities?

  • Approach: Develop antibody-drug conjugates targeting IARS2 and assess selectivity between cancer cells and normal tissues, based on findings that IARS2 knockdown induces apoptosis in melanoma cells .

• Interorganelle communication:

  • Research question: Does IARS2 participate in signaling between mitochondria and nucleus during mitochondrial dysfunction?

  • Approach: Subcellular fractionation with IARS2 antibodies to track potential translocation between compartments during retrograde signaling, combined with proximity ligation assays to identify dynamic interaction partners.