NDUFA13 Antibody

What is NDUFA13 and what is its biological significance?

NDUFA13 (NADH:ubiquinone oxidoreductase subunit A13) is an accessory subunit of mitochondrial respiratory chain Complex I. In humans, it is a 144 amino acid protein with a molecular weight of approximately 16.7 kDa . NDUFA13 is also known by several other names including GRIM-19, CGI-39, and B16.6 .

The protein has significant biological importance as it contains a unique transmembrane helix (TMH) structure that can penetrate both Iα and Iλ structures within complex I . This structural characteristic is thought to be critical for maintaining mitochondrial membrane integrity. NDUFA13 is widely expressed across tissues, with notably high expression in metabolically active organs such as the heart, skeletal muscle, liver, kidney, and placenta . Beyond its role in the respiratory chain, NDUFA13 has been implicated in apoptotic signaling and ROS generation, making it relevant to both normal cellular physiology and disease states .

How does NDUFA13 function within mitochondrial Complex I?

What experimental methods are commonly used to detect NDUFA13?

Detection of NDUFA13 in research settings typically employs several complementary techniques:

• Western Blotting: The most common method for quantifying NDUFA13 protein levels, typically using 10-12% SDS-PAGE gels with PVDF membrane transfer . Researchers often use commercially available antibodies against NDUFA13, with β-actin, α-tubulin, or VDAC (for mitochondrial fractions) serving as loading controls.

• Immunofluorescence: For localization studies, particularly to confirm mitochondrial positioning of NDUFA13 or its mutant variants. This technique can verify colocalization with mitochondrial markers .

• Subcellular Fractionation: To separate mitochondrial and cytoplasmic protein fractions when studying NDUFA13's specific localization and function. Commercial mitochondria isolation kits can be employed for this purpose .

• Flow Cytometry: For assessing mitochondrial membrane potential in conjunction with NDUFA13 manipulation, using probes such as TMRM (tetramethylrhodamine methyl ester) .

How does NDUFA13 modulation affect ROS generation in mitochondria?

NDUFA13 plays a nuanced role in ROS generation, with effects that vary based on the degree of expression modulation and cellular context. Research using cardiac-specific NDUFA13 heterozygous knockout (cHet) mice has revealed several important characteristics:

• Moderate NDUFA13 downregulation (as in cHet mice) creates an electron leak within Complex I that results in increased cytoplasmic H₂O₂ levels, but not superoxide, under basal conditions . This H₂O₂ appears to function as a second messenger that activates cytoprotective pathways.

• Spatial distribution of ROS: Using targeted H₂O₂ sensors (cyto-HyPer for cytoplasm and mito-HyPer for mitochondria), researchers demonstrated that NDUFA13 downregulation specifically increases cytosolic H₂O₂ without affecting mitochondrial H₂O₂ levels at baseline . This suggests a directed release of ROS from complex I to the cytosol.

• Effect on reverse electron transport (RET): Mitochondria from cHet mice exhibited significantly reduced RET-induced H₂O₂ generation when exposed to succinate, indicating an interrupted RET process . This represents an important mechanism through which NDUFA13 modulation may protect against ischemia-reperfusion injury.

• Response to stress conditions: During hypoxia/reoxygenation or ischemia/reperfusion, NDUFA13-depleted cells show significantly reduced superoxide burst compared to control cells . This suggests that moderate NDUFA13 downregulation establishes a preconditioning-like state that prevents excessive ROS generation during stress.

These findings indicate that NDUFA13 serves as a regulatory point for controlled ROS generation that may have important implications for cardioprotection and cellular adaptation to stress.

What is the relationship between NDUFA13 and apoptotic signaling pathways?

NDUFA13 exhibits a complex relationship with apoptotic signaling that appears to be dose-dependent:

• Moderate downregulation of NDUFA13 (approximately 30% decrease) confers protection against apoptosis in cardiomyocytes exposed to hypoxia/reoxygenation injury . This is evidenced by decreased TUNEL-positive cells, reduced cleaved caspase-3 and caspase-9 expression, and diminished cytochrome c release into the cytosol.

• Severe downregulation (approximately 60% decrease) fails to elicit protection and may actually impair mitochondrial membrane potential . This suggests an optimal window of NDUFA13 expression for cytoprotection.

• Intrinsic vs. extrinsic apoptotic pathways: NDUFA13 modulation primarily affects the intrinsic mitochondrial apoptotic pathway, as evidenced by changes in cleaved caspase-9 and cytochrome c release, while markers of the extrinsic pathway such as cleaved caspase-8 remain unaffected .

• STAT3 signaling: Decreased NDUFA13 expression leads to STAT3 dimerization and activation of antiapoptotic signaling, suggesting a mechanistic link between NDUFA13, ROS signaling, and apoptotic regulation . This appears to be mediated by the mild increase in cytoplasmic H₂O₂ that occurs with moderate NDUFA13 downregulation.

• Tumor context: In cancer research, decreased NDUFA13 expression has been associated with enhanced resistance to apoptosis in tumor cells . This suggests contextual differences in how NDUFA13 modulation affects apoptosis in different cell types or disease states.

These findings collectively suggest that NDUFA13 serves as a regulatory node connecting mitochondrial function, ROS signaling, and apoptotic pathways, with potential implications for both cardioprotection and cancer biology.

What experimental models are available for studying NDUFA13 function?

Several experimental models have been developed to investigate NDUFA13 function:

• Cardiac-specific conditional knockout mice:

  • Cardiac-specific tamoxifen-inducible NDUFA13 knockout models (Myh6Cre⁺NDUFA13ᶠˡᵒˣ/ᶠˡᵒˣ for homozygous deletion and Myh6Cre⁺NDUFA13ᶠˡᵒˣ/⁻ for heterozygous deletion)

  • These models allow for temporal control of NDUFA13 deletion specifically in cardiomyocytes, enabling investigation of both acute and chronic effects

• Cellular models:

  • siRNA-mediated knockdown in H9C2 cells (cardiac cell line)

  • Neonatal mouse cardiomyocytes (NMCMs) isolated from NDUFA13ᶠˡᵒˣ/⁻ or NDUFA13ᶠˡᵒˣ/ᶠˡᵒˣ mice and transfected with adenovirus containing Cre recombinase

  • Rescue experiments using adenoviral vectors expressing wild-type or truncated NDUFA13 mutants

• Stress models:

  • In vitro hypoxia/reoxygenation (H/R) model: typically 6 hours of hypoxia followed by 18 hours of reoxygenation

  • In vivo ischemia/reperfusion (I/R) model: coronary artery ligation for 45 minutes followed by 3 hours of reperfusion

• ROS detection systems:

  • Targeted ROS sensors: cytoplasm-targeting HyPer (cyto-HyPer) and mitochondrial-targeting HyPer (mito-HyPer) for specific H₂O₂ detection

  • MitoSOX Red for mitochondrial superoxide detection

  • Oroboros O2k system for simultaneous measurement of oxygen consumption rate (OCR) and H₂O₂ levels in isolated mitochondria

These models provide complementary approaches for investigating NDUFA13 function at molecular, cellular, and organismal levels, enabling researchers to address questions ranging from basic protein structure-function relationships to physiological impacts in intact animals.

How does the structure of NDUFA13 relate to its function?

NDUFA13 possesses unique structural features that are critical to its function within Complex I:

• Transmembrane helix (TMH): NDUFA13 is the only protein in Complex I containing a transmembrane helix that penetrates both Iα and Iλ structures within the complex . This unique structural characteristic appears essential for proper integration into the mitochondrial membrane.

• Structure-function experiments: Research using truncated NDUFA13 mutants has identified critical regions:

  • Deletion of amino acids 40-50 (Ad-1 mutant) prevented proper mitochondrial localization and failed to maintain mitochondrial membrane potential

  • Deletions in other regions (amino acids 70-80 or 110-120) did not affect localization or function

  • This indicates the 40-50 amino acid region containing the TMH is essential for NDUFA13's proper integration and function

• Proximity to low electrochemical potential regions: Within Complex I, NDUFA13 is positioned close to segments with lower electrochemical potentials . This strategic location may explain how moderate downregulation creates a controlled electron leak that generates cytoprotective levels of ROS.

• Interaction with other Complex I components: While not explicitly detailed in the search results, NDUFA13's position within Complex I suggests interactions with other subunits that may be critical for maintaining the structural integrity and electron flow through the complex.

Understanding these structural characteristics is essential for interpreting how NDUFA13 modulation affects mitochondrial function, ROS generation, and downstream signaling pathways.

What are the best practices for detecting NDUFA13 using Western blotting?

When conducting Western blot analysis for NDUFA13, researchers should consider the following methodological details:

• Sample preparation:

  • For whole cell or tissue lysates: Extract proteins in RIPA solution containing protease inhibitor mixture

  • For subcellular fractionation: Use a mitochondria isolation kit to separate mitochondrial and cytoplasmic fractions

  • Quantify protein using BCA Protein Assay Kit

• Gel electrophoresis and transfer:

  • Load approximately 10 μg of protein on 12% SDS-PAGE gel

  • Transfer to PVDF membrane

  • Ensure complete transfer of lower molecular weight proteins (NDUFA13 is approximately 16.7 kDa)

• Antibody selection and validation:

  • Use validated anti-NDUFA13 antibodies (commercially available from multiple vendors)

  • Include appropriate positive controls (tissues with high NDUFA13 expression include heart, skeletal muscle, liver, kidney)

  • Confirm antibody specificity using knockout/knockdown controls when possible

• Loading controls:

  • For whole cell lysates: β-Actin or α-Tubulin

  • For mitochondrial fractions: VDAC or other mitochondrial markers like ATP5A

  • For cytoplasmic fractions: α-Tubulin or other cytoplasmic markers

• Quantification:

  • Use ImageLab or similar software for densitometric analysis

  • Normalize NDUFA13 signal to appropriate loading controls

  • Present data as fold-change relative to control conditions

These methodological considerations help ensure reliable detection and quantification of NDUFA13 protein levels in various experimental contexts.

How can researchers generate and validate NDUFA13 knockdown models?

Researchers have employed several strategies to generate NDUFA13 knockdown models:

• siRNA-mediated knockdown:

  • Transfect cells with NDUFA13-targeting siRNA (e.g., sequence 5′GCCUUGAUCUUUGGCUACUTT3′ for rat NDUFA13)

  • Optimize siRNA concentration to achieve desired level of knockdown (moderate vs. severe)

  • Use Lipofectamine 2000 or similar transfection reagent in serum-free, antibiotic-free medium

  • Change medium 6 hours post-transfection

  • Validate knockdown efficiency 24-48 hours after transfection by Western blot

• Conditional knockout mouse models:

  • Generate mice with floxed NDUFA13 alleles (NDUFA13ᶠˡᵒˣ/ᶠˡᵒˣ)

  • Cross with tissue-specific Cre-expressing mice (e.g., Myh6Cre for cardiac-specific deletion)

  • For inducible models, use tamoxifen-inducible Cre systems

  • Monitor NDUFA13 expression at multiple timepoints after induction (e.g., days 1, 4, 7, 10, 13, 16)

  • Validate knockout efficiency by Western blot analysis of target tissue

• Adenoviral Cre delivery:

  • Isolate primary cells (e.g., neonatal cardiomyocytes) from NDUFA13ᶠˡᵒˣ/ᶠˡᵒˣ or NDUFA13ᶠˡᵒˣ/⁻ mice

  • Transfect with adenovirus containing Cre recombinase (Ad-Cre)

  • Use empty vector adenovirus (Ad-NC) as control

  • Validate knockdown efficiency by Western blot

• Validation approaches:

  • Protein level: Western blot analysis using anti-NDUFA13 antibodies

  • Functional validation: Assess mitochondrial membrane potential using TMRM staining

  • Subcellular localization: Immunofluorescence to confirm mitochondrial localization

  • Phenotypic validation: Measure parameters like ATP levels, oxygen consumption rate, or response to stress conditions

Importantly, the degree of NDUFA13 knockdown significantly impacts cellular phenotypes, with moderate reduction (approximately 30%) offering protection against stress conditions while severe reduction (approximately 60%) potentially impairing mitochondrial function . Researchers should carefully titrate their knockdown approaches to achieve the desired level of NDUFA13 reduction.

What methods should be used to assess ROS generation in NDUFA13-related experiments?

When investigating ROS production in the context of NDUFA13 modulation, researchers should employ multiple complementary approaches to capture different ROS species and their subcellular localization:

• Targeted H₂O₂ detection:

  • Cytoplasmic H₂O₂: Use adenovirus containing cytoplasm-targeting HyPer (cyto-HyPer)

  • Mitochondrial H₂O₂: Use adenovirus containing mitochondrial-targeting HyPer (mito-HyPer)

  • These genetically-encoded sensors provide compartment-specific H₂O₂ detection with high sensitivity

• Superoxide detection:

  • Mitochondrial superoxide: Use MitoSOX Red, a mitochondria-targeted probe that fluoresces upon oxidation by superoxide

  • Analyze by fluorescence microscopy or flow cytometry

  • Important for distinguishing between superoxide and H₂O₂ generation

• Combined OCR and H₂O₂ measurement:

  • Use the Oroboros O2k system to simultaneously measure oxygen consumption rate and H₂O₂ levels in isolated mitochondria

  • This allows correlation between electron transport chain activity and ROS production

  • Apply specific substrates and inhibitors to identify ROS sources:

    • Succinate: To induce reverse electron transport (RET)

    • Rotenone: To block Complex I

    • Pyruvate and malate: Complex I substrates

    • Antimycin A: Complex III inhibitor

• Experimental design considerations:

  • Basal vs. stress conditions: Assess ROS generation both under basal conditions and during stress (e.g., hypoxia/reoxygenation, ischemia/reperfusion)

  • Time course analysis: Monitor ROS generation at multiple timepoints to capture dynamic changes

  • Controls: Include appropriate positive controls (e.g., antimycin A treatment) and negative controls (e.g., antioxidant treatment)

  • Validation: Confirm ROS findings using multiple detection methods whenever possible

This multi-faceted approach allows researchers to comprehensively characterize the complex ROS profile associated with NDUFA13 modulation, including species specificity (H₂O₂ vs. superoxide), subcellular localization, and dynamic changes under different conditions.

How should mitochondrial isolation be performed for NDUFA13 functional studies?

Proper mitochondrial isolation is critical for studying NDUFA13 function, as this protein is primarily localized to mitochondria and plays a key role in Complex I. The following methodological approach is recommended:

• Tissue preparation:

  • For mouse heart tissue: Rapidly excise the heart and place in ice-cold isolation buffer

  • Rinse thoroughly to remove blood

  • Mince tissue into small pieces in isolation buffer

• Mitochondrial isolation:

  • Use a commercial Mitochondria Isolation Kit according to the manufacturer's instructions

  • Alternatively, employ differential centrifugation techniques:

    • Homogenize tissue in isolation buffer (typically containing sucrose, HEPES, EGTA, and BSA)

    • Centrifuge at low speed (800-1000g) to remove nuclei and debris

    • Centrifuge supernatant at higher speed (8000-10000g) to pellet mitochondria

    • Wash mitochondrial pellet to remove contaminants

• Quality assessment:

  • Verify mitochondrial enrichment by Western blot using mitochondrial markers (e.g., VDAC, ATP5A)

  • Confirm depletion of cytoplasmic markers (e.g., α-Tubulin)

  • Assess mitochondrial membrane integrity using membrane potential dyes like TMRM

  • Measure oxygen consumption rate to confirm functional integrity

• Functional assays with isolated mitochondria:

  • Oxygen consumption: Use platforms like Oroboros O2k to measure respiratory capacity

  • ROS generation: Simultaneously measure H₂O₂ production alongside oxygen consumption

  • Substrate utilization: Test different substrates (pyruvate/malate for Complex I, succinate for Complex II, TMPD+ascorbate for Complex IV)

  • Response to inhibitors: Assess sensitivity to rotenone (Complex I inhibitor) and antimycin A (Complex III inhibitor)

• Storage considerations:

  • Fresh isolation is preferred for functional studies

  • If storage is necessary, snap-freeze mitochondrial pellets in liquid nitrogen

  • Store at -80°C for protein analysis

  • Note that freeze-thaw cycles may compromise functional integrity

These methodological considerations ensure reliable isolation of functional mitochondria for comprehensive analysis of NDUFA13's role in mitochondrial physiology and ROS generation.

How can NDUFA13 antibodies be used to study ischemia-reperfusion injury mechanisms?

NDUFA13 antibodies serve as valuable tools for investigating mechanisms of ischemia-reperfusion (I/R) injury, particularly in cardiac tissue where NDUFA13 modulation has demonstrated protective effects:

• Protein expression analysis:

  • Use anti-NDUFA13 antibodies to quantify expression levels in tissues exposed to I/R injury

  • Compare NDUFA13 expression between ischemic, peri-infarct, and remote zones

  • Correlate NDUFA13 levels with markers of cell death and oxidative stress

• Subcellular redistribution:

  • Employ immunofluorescence with anti-NDUFA13 antibodies to track potential subcellular redistribution during I/R

  • Co-stain with mitochondrial markers to assess mitochondrial localization

  • Examine potential nuclear translocation, as NDUFA13 has been reported to localize to both mitochondria and nucleus

• Protein-protein interactions:

  • Use anti-NDUFA13 antibodies for co-immunoprecipitation studies to identify interaction partners during I/R

  • Investigate potential interactions with STAT3, as NDUFA13 downregulation activates STAT3 signaling

  • Examine interactions with other Complex I subunits to assess structural integrity during stress

• Experimental design for I/R studies:

  • In the in vivo cardiac I/R model, perform coronary artery ligation for 45 minutes followed by 3 hours of reperfusion

  • Collect tissue from infarct, peri-infarct, and remote zones for Western blot analysis

  • Use TUNEL staining to quantify apoptosis, particularly in the peri-infarct area

  • Measure mitochondrial parameters including cytochrome c release and ROS generation

Research has demonstrated that moderate NDUFA13 downregulation protects against I/R injury through mechanisms involving increased basal cytosolic H₂O₂, STAT3 activation, and suppression of apoptosis . NDUFA13 antibodies are essential tools for elucidating these protective mechanisms and potentially identifying therapeutic targets for ischemic diseases.

What approaches can be used to study the interaction between NDUFA13 and STAT3 signaling?

The interaction between NDUFA13 and STAT3 signaling represents a critical node connecting mitochondrial function to cellular survival pathways. Researchers can investigate this relationship using several complementary approaches:

• Activation state analysis:

  • Use Western blotting with anti-STAT3 antibodies to assess total STAT3 levels

  • Examine STAT3 dimerization through non-reducing SDS-PAGE followed by Western blotting

  • Investigate phosphorylation status of STAT3 at key regulatory sites

• Nuclear translocation:

  • Perform subcellular fractionation to isolate nuclear and cytoplasmic fractions

  • Quantify nuclear STAT3 levels by Western blotting

  • Use immunofluorescence to visualize STAT3 nuclear translocation

• Downstream signaling assessment:

  • Measure expression of STAT3-regulated genes involved in anti-apoptotic pathways

  • Quantify levels of anti-apoptotic proteins like Bcl-2

  • Assess activation of apoptotic pathways (cleaved caspase-3, caspase-9)

• Causal relationship studies:

  • Use STAT3 inhibitors in NDUFA13-downregulated models to confirm the role of STAT3 in observed protective effects

  • Employ ROS scavengers to determine if H₂O₂ serves as an intermediary between NDUFA13 downregulation and STAT3 activation

  • Perform gain-of-function experiments with constitutively active STAT3 to mimic effects of NDUFA13 downregulation

• Protein-protein interaction analysis:

  • Conduct co-immunoprecipitation experiments using anti-NDUFA13 or anti-STAT3 antibodies

  • Perform proximity ligation assays to detect potential direct interactions

  • Use subcellular fractionation to determine compartment-specific interactions

Research has demonstrated that moderate NDUFA13 downregulation leads to increased cytosolic H₂O₂, which serves as a second messenger that promotes STAT3 dimerization and activation of anti-apoptotic signaling . This pathway appears central to the cardioprotective effects observed in NDUFA13 heterozygous knockout mice during ischemia-reperfusion injury .

What are common challenges when using NDUFA13 antibodies and how can they be addressed?

When working with NDUFA13 antibodies, researchers may encounter several technical challenges that can impact experimental outcomes:

• Multiple protein isoforms:

  • Challenge: Up to two different isoforms of NDUFA13 have been reported , which may complicate band interpretation

  • Solution: Use positive controls with known expression patterns to identify specific isoforms

  • Approach: Compare band patterns across multiple tissues with differential isoform expression

• Low molecular weight detection:

  • Challenge: NDUFA13 is a relatively small protein (16.7 kDa) , which can be difficult to resolve and transfer

  • Solution: Use higher percentage gels (12-15%) for better resolution of low molecular weight proteins

  • Approach: Optimize transfer conditions (lower voltage, longer time) for small proteins

• Subcellular localization:

  • Challenge: NDUFA13 localizes to both mitochondria and nucleus , requiring careful interpretation

  • Solution: Perform proper subcellular fractionation to distinguish between compartments

  • Approach: Include compartment-specific markers (VDAC for mitochondria, nuclear lamin for nucleus)

• Specificity verification:

  • Challenge: Ensuring antibody specificity against the target protein

  • Solution: Include appropriate knockdown/knockout controls

  • Approach: Compare results from multiple antibodies targeting different epitopes of NDUFA13

• Cross-reactivity across species:

  • Challenge: NDUFA13 orthologs exist in multiple species including mouse, rat, bovine, frog, zebrafish, chimpanzee, and chicken

  • Solution: Verify antibody specificity for the species being studied

  • Approach: Test antibody performance using positive controls from the relevant species

• Quantification accuracy:

  • Challenge: Accurate normalization when NDUFA13 expression is experimentally manipulated

  • Solution: Use multiple loading controls appropriate for the subcellular fraction being analyzed

  • Approach: Normalize to total protein staining methods like Ponceau S in addition to specific loading controls

How can researchers distinguish between direct effects of NDUFA13 modulation and secondary consequences?

Distinguishing primary from secondary effects of NDUFA13 modulation presents a significant challenge in research. The following methodological approaches can help researchers delineate direct consequences from downstream effects:

• Temporal analysis:

  • Monitor changes in multiple parameters at different time points after NDUFA13 modulation

  • In inducible models, track the sequence of events following tamoxifen administration

  • Early changes (days 1-4) are more likely to represent direct effects, while later changes (days 10-16) may reflect adaptive or compensatory responses

• Dose-response relationships:

  • Compare effects of different degrees of NDUFA13 downregulation (e.g., 30% vs. 60%)

  • Determine if there is a threshold effect or linear relationship between NDUFA13 levels and functional outcomes

  • Different phenotypes at different expression levels may reveal distinct mechanisms

• Rescue experiments:

  • Re-express wild-type NDUFA13 in knockout/knockdown models to confirm direct causality

  • Use structure-function analysis with truncated NDUFA13 mutants to identify critical domains

  • Direct effects should be reversible with reconstitution of wild-type protein

• Pathway inhibitors:

  • Use specific inhibitors to block potential intermediate signaling pathways

  • For example, ROS scavengers can determine if observed effects depend on H₂O₂ generation

  • STAT3 inhibitors can confirm the role of this pathway in mediating protective effects

• Domain-specific mutants:

  • Engineer domain-specific mutations that affect particular functions of NDUFA13

  • Compare phenotypes between transmembrane helix mutants (Ad-1) and other domain mutants (Ad-2, Ad-3)

  • This approach can separate structural roles from signaling functions

• Compartment-specific measurements:

  • Assess parameters in specific subcellular compartments (e.g., cytosolic vs. mitochondrial H₂O₂)

  • Direct effects of NDUFA13 modulation should manifest first in compartments where it normally resides

Research has demonstrated that moderate NDUFA13 downregulation creates a controlled electron leak in Complex I that increases cytoplasmic H₂O₂, which then acts as a second messenger to activate STAT3 signaling . This represents a primary-to-secondary effect cascade that links mitochondrial function to nuclear signaling and ultimately to cellular protection against apoptosis.