FOXRED1 Antibody

What is FOXRED1 and why is it significant in mitochondrial research?

FOXRED1 (FAD-dependent oxidoreductase domain-containing protein 1) is a 486 amino acid protein with an approximate mass of 53 kDa that contains an FAD-dependent oxidoreductase domain. It functions as a molecular chaperone required for the assembly, stability, and proper functioning of mitochondrial respiratory chain NADH dehydrogenase (Complex I) . The protein is particularly significant because mutations in the FOXRED1 gene are associated with mitochondrial complex I deficiency, which can present as severe multisystem disorders . FOXRED1 is involved specifically in the mid-late stages of complex I assembly and its dysfunction has been linked to mitochondrial DNA depletion syndrome where it may interact with other mitochondrial proteins contributing to pathological phenotypes .

Researchers studying mitochondrial disorders, neurological conditions, or energy metabolism should consider FOXRED1 a valuable target due to its essential role in oxidative phosphorylation and its involvement in diseases with complex clinical presentations.

What are the optimal applications for FOXRED1 antibodies in research?

FOXRED1 antibodies have been validated for several key applications in mitochondrial research:

For Western blot applications, researchers should be aware that while the calculated molecular weight of FOXRED1 is 54 kDa, observed molecular weights can be 30 kDa and 50 kDa, likely representing different isoforms or processed forms of the protein . When designing experiments, it's recommended to use positive controls such as extracts from cells known to express FOXRED1 (like HeLa or HepG2) to establish appropriate detection conditions.

For immunohistochemistry, antigen retrieval methods significantly impact results, with TE buffer pH 9.0 showing better performance than citrate buffer in preserving epitope recognition .

How do FOXRED1 expression patterns vary across tissues and cell types?

FOXRED1 shows a tissue-specific expression pattern that correlates with energy demands. The protein demonstrates significant expression in tissues with high metabolic requirements, particularly:

• Brain tissue (high expression)

• Muscle tissue (high expression)

• Various cell lines including HeLa, HepG2, A549, A431, and Jurkat

This expression pattern reflects FOXRED1's critical role in mitochondrial function and energy production. When designing experiments to study FOXRED1, consider that expression levels may vary significantly between tissues and cell types, necessitating optimization of antibody dilutions and detection methods for each experimental system.

Steady-state mRNA transcripts for FOXRED1 have been detected in at least 12 human tissues, suggesting widespread expression similar to other previously identified complex I assembly factors . Bioinformatics analysis using mouse tissue atlas data shows a strong positive correlation between FOXRED1 expression and expression of known complex I subunits, further supporting its functional association with the complex .

What controls should be included when using FOXRED1 antibodies?

Proper controls are essential for generating reliable results with FOXRED1 antibodies:

Research has demonstrated that stable knockdown of FOXRED1 expression via lentiviral-mediated shRNAi reduces complex I expression (as assessed with anti-NDUFA9 antibody) . These FOXRED1-silenced cells provide excellent negative controls to validate antibody specificity. Additionally, complementation studies with wild-type FOXRED1 in patient fibroblasts show restoration of protein levels detectable by Western blotting, providing another system to validate antibody performance .

When investigating suspected pathogenic variants, researchers should include both mutant and wild-type FOXRED1-expressing cells when possible, as this allows for direct comparison of antibody recognition and protein expression levels under identical experimental conditions.

What are the technical considerations for optimizing Western blots with FOXRED1 antibodies?

Optimization of Western blot protocols for FOXRED1 detection requires attention to several key parameters:

• Sample preparation: Mitochondrial fractions typically yield cleaner results than whole cell lysates due to FOXRED1's mitochondrial localization . Consider using mitochondrial isolation protocols that preserve protein integrity.

• Gel percentage: 10% SDS-PAGE gels have been successfully used for separating FOXRED1 . For higher resolution of the 30 kDa and 50 kDa forms, 12% gels may be preferable.

• Transfer conditions: Semi-dry transfer systems with PVDF membranes have shown good results for FOXRED1 detection.

• Blocking agent: 5% non-fat milk in TBST is typically effective, but BSA-based blockers may reduce background in some applications.

• Detection of multiple bands: Be prepared to observe both the 50 kDa full-length protein and a 30 kDa form, which may represent a processed form or alternative splice variant .

• Stripping and reprobing: When analyzing complex I assembly in the same samples, membranes can be stripped and reprobed with antibodies against other complex I subunits like NDUFA9 .

For Blue Native PAGE (BN-PAGE) analysis, which is particularly valuable for studying complex I assembly, solubilize native mitochondrial complexes with 2% digitonin before separation on gradient gels (3-12% or 4-16%) . This approach can reveal assembly intermediates and allows assessment of holoenzyme levels.

How can FOXRED1 antibodies be used to investigate mitochondrial complex I deficiencies?

FOXRED1 antibodies are valuable tools for investigating complex I deficiencies through several methodological approaches:

• Assessment of steady-state FOXRED1 levels: Western blot analysis can reveal reduced FOXRED1 protein in patient fibroblasts carrying pathogenic variants, as demonstrated in previous studies . This provides a direct measure of the impact of mutations on protein stability.

• Complex I holoenzyme quantification: BN-PAGE followed by immunoblotting can assess the impact of FOXRED1 deficiency on complex I assembly. Patient fibroblasts with FOXRED1 mutations show marked reduction in steady-state levels of complex I holoenzyme compared to control cell lines .

• Functional rescue experiments: Lentiviral-mediated complementation with wild-type FOXRED1 cDNA in patient fibroblasts can restore both FOXRED1 protein levels and complex I activity, confirming the causative role of FOXRED1 variants in complex I deficiency . Western blotting after complementation allows visualization of restored protein expression.

• Correlation with enzymatic activity: Combine antibody-based detection of FOXRED1 and complex I subunits with activity assays (spectrophotometric or dipstick) to correlate protein levels with functional deficits. In previous studies, patient fibroblasts showed ∼70% residual complex I activity relative to controls, which increased to ∼90% after FOXRED1 complementation .

These approaches allow researchers to establish mechanistic links between FOXRED1 variants and complex I dysfunction, providing insights into pathogenicity mechanisms.

How can researchers utilize FOXRED1 antibodies to study protein-protein interactions within complex I assembly?

Investigating FOXRED1's interactions with other complex I components requires sophisticated approaches combining antibody-based detection with interaction studies:

• Co-immunoprecipitation (Co-IP): FOXRED1 antibodies can be used to pull down FOXRED1 and its interacting partners from mitochondrial lysates. This approach can identify both stable and transient interactions with complex I subunits and other assembly factors.

• Proximity ligation assay (PLA): This technique combines antibody recognition with DNA amplification to visualize protein interactions in situ. Using FOXRED1 antibodies in combination with antibodies against putative interaction partners allows direct visualization of protein complexes within mitochondria.

• BN-PAGE followed by second-dimension SDS-PAGE: This technique separates intact complexes in the first dimension, then individual proteins in the second dimension. Immunoblotting with FOXRED1 antibodies can identify which complex I assembly intermediates contain FOXRED1.

• Cross-linking mass spectrometry: Chemical cross-linking followed by immunoprecipitation with FOXRED1 antibodies and mass spectrometry analysis can map interaction interfaces at the amino acid level.

The strong positive correlation between FOXRED1 expression and expression of known complex I subunits, as revealed by bioinformatics analysis, implies functional associations that should be experimentally validated . Such studies would benefit from FOXRED1 antibodies used in combination with antibodies against other complex I components.

What approaches can resolve contradictory results when assessing FOXRED1 expression with different antibodies?

Contradictory results with different FOXRED1 antibodies can be systematically addressed through:

• Epitope mapping: Compare the immunogens used to generate each antibody. The Proteintech FOXRED1 antibody (24595-1-AP) was raised against a fusion protein antigen , while the Abcam antibody (ab229860) targets a recombinant fragment within amino acids 300 to C-terminus . Different epitopes may be differentially accessible in certain experimental conditions.

• Multiple detection methods: Combine Western blotting with immunofluorescence or mass spectrometry-based approaches to obtain concordant evidence of expression patterns.

• Validation in knockout/knockdown models: Test antibodies in FOXRED1-depleted samples. Previous studies have shown that stable knockdown of FOXRED1 expression via lentiviral-mediated shRNAi reduces protein detection by Western blot .

• Comparison across fixation and extraction methods: Different extraction buffers and fixation protocols can affect epitope accessibility. Systematic comparison can identify optimal conditions for each antibody.

• Isoform-specific detection: The observed molecular weights of 30 kDa and 50 kDa suggest possible isoforms or processed forms of FOXRED1 . Different antibodies may preferentially detect specific forms depending on epitope location.

When contradictory results persist, complementary approaches like RNA-seq or qPCR can provide transcript-level data to support protein expression findings.

How can FOXRED1 antibodies contribute to understanding the relationship between genotype and phenotype in mitochondrial disorders?

FOXRED1 antibodies offer powerful tools for exploring genotype-phenotype correlations in mitochondrial disease:

• Quantitative expression analysis: Western blot analysis using FOXRED1 antibodies can quantify residual protein levels in patient samples with different pathogenic variants. This data can be correlated with clinical severity to establish relationships between protein levels and phenotype.

• Tissue-specific expression patterns: Immunohistochemistry with FOXRED1 antibodies can reveal differential expression patterns across tissues, potentially explaining tissue-specific manifestations of FOXRED1 mutations.

• Functional complementation studies: The impact of different FOXRED1 variants can be assessed by expressing mutant proteins in deficient cells and measuring protein levels (via Western blot) and complex I activity restoration. Previous studies showed that lentiviral-mediated expression of synthetic FOXRED1 in patient fibroblasts rescued complex I activity to ~90% of control levels .

• Structure-function analysis: Combining biochemical data with in silico protein modeling can reveal how specific mutations affect FOXRED1 structure and function. For example, the R352W mutation has been predicted to impinge on the FAD-binding site, potentially interfering with FAD binding .

The clinical spectrum associated with FOXRED1 defects is expanding as new variants are characterized . Patients have presented with conditions ranging from infantile-onset encephalomyopathy to ataxia, epilepsy, and psychomotor developmental delay . Correlating these phenotypes with biochemical and cellular findings using FOXRED1 antibodies can illuminate pathogenic mechanisms and potentially guide therapeutic approaches.

How can FOXRED1 antibodies be employed in studying post-translational modifications?

FOXRED1 contains an FAD-dependent oxidoreductase domain, suggesting potential regulation through post-translational modifications (PTMs). Researchers can investigate PTMs using:

• Phospho-specific antibodies: While not commercially available yet, custom antibodies against predicted phosphorylation sites could reveal regulation of FOXRED1 activity.

• Two-dimensional gel electrophoresis: Combining isoelectric focusing with SDS-PAGE followed by FOXRED1 immunoblotting can separate differentially modified forms of the protein.

• Immunoprecipitation and mass spectrometry: FOXRED1 antibodies can be used to pull down the protein from cellular lysates, followed by mass spectrometry analysis to identify PTMs comprehensively.

• PTM-specific staining: After immunoprecipitation with FOXRED1 antibodies, Western blots can be probed with PTM-specific stains (ProQ Diamond for phosphorylation, etc.) to detect modifications.

The FAD-binding properties of FOXRED1 suggest that redox-dependent modifications might regulate its function. In silico modeling indicates that the R352W mutation could affect the FAD-binding site , highlighting the importance of this domain for protein function.

What role might FOXRED1 play in cellular responses to metabolic stress and how can antibodies help investigate this?

FOXRED1's role in mitochondrial function suggests it may participate in cellular responses to metabolic stress. Research approaches using FOXRED1 antibodies might include:

• Stress-response profiling: Western blot analysis of FOXRED1 expression under various metabolic stressors (hypoxia, nutrient deprivation, etc.) can reveal regulatory patterns.

• Subcellular localization studies: Immunofluorescence with FOXRED1 antibodies can track potential redistribution of the protein under stress conditions.

• Interaction dynamics: Co-immunoprecipitation with FOXRED1 antibodies under normal and stress conditions can identify stress-specific interaction partners.

• Degradation kinetics: Pulse-chase experiments combined with immunoprecipitation can assess protein stability under different metabolic conditions.

FOXRED1 shows significant expression in tissues with high energy demand such as muscle and brain , suggesting it may play critical roles in tissues that are particularly vulnerable to metabolic stress. Understanding how FOXRED1 responds to such stressors could provide insights into tissue-specific pathologies in mitochondrial diseases.