FBXO10 Antibody, FITC conjugated

What is FBXO10 and why is it significant in mitochondrial research?

FBXO10 is an F-box protein that functions as a substrate recognition component of SCF (SKP1-CUL1-F-box) E3 ubiquitin ligase complexes. Recent research demonstrates that FBXO10 localizes to the outer mitochondrial membrane (OMM) where it regulates selective protein turnover essential for mitochondrial homeostasis. FBXO10 forms an authentic CRL1 (Cullin-RING ligase 1) complex at mitochondria, as evidenced by co-immunoprecipitation of SKP1 and CUL1 from enriched mitochondrial fractions .

Significance stems from its role in mitochondrial quality control through ubiquitin-mediated degradation of specific OMM proteins. Unbiased quantitative mass spectrometry has revealed at least eighteen OMM and/or OMM-associated protein targets showing significant reciprocal changes in protein levels when comparing wild-type FBXO10 versus the CaaX-mutant FBXO10(C953S) . This regulatory function impacts mitochondrial ATP production, membrane potential, and morphological dynamics, making FBXO10 a critical component in mitochondrial proteostasis research.

What applications are most suitable for FITC-conjugated FBXO10 antibodies?

FITC-conjugated FBXO10 antibodies are particularly valuable for:

• Flow cytometry-based quantification of FBXO10 in intact mitochondrial fractions, allowing for precise measurement of mitochondrial association as demonstrated in subcellular localization studies

• Live-cell confocal microscopy to visualize FBXO10's dynamic subcellular distribution, especially in response to pharmacological interventions such as geranylgeranylation inhibitors or HSP90 inhibitors

• Immunofluorescence microscopy for analyzing subcellular distribution patterns of FBXO10, specifically distinguishing between mitochondrial localization and homogeneous cytosolic distribution

The direct fluorescent labeling eliminates the need for secondary antibodies, reducing background and cross-reactivity issues that can complicate interpretation of mitochondrial protein localization studies.

How can I validate the specificity of FBXO10 antibodies in mitochondrial research?

Validating FBXO10 antibody specificity requires a multi-faceted approach:

• CRISPR-Cas9 knockout controls: Generate FBXO10 knockout cell lines as negative controls. Complete absence of signal in knockout cells confirms antibody specificity .

• Subcellular fractionation validation: Compare signals between mitochondrial, cytosolic, and whole cell lysate fractions. True FBXO10 antibodies should show enrichment in mitochondrial fractions consistent with the known localization pattern of endogenous FBXO10 .

• Differential detection of mutants: Test the antibody against FBXO10(C953S) mutant, which displays altered subcellular distribution. Proper antibodies should detect both wild-type and mutant forms while revealing their distinct localization patterns .

• Cross-validation with tagged constructs: Compare antibody staining patterns with fluorescently-tagged FBXO10 constructs (such as GFP-FBXO10 or ptd-Tomato-FBXO10) to ensure concordance in localization patterns .

• Western blot molecular weight verification: Confirm that the detected protein band corresponds to the predicted molecular weight of FBXO10 (approximately 65 kDa) .

What is the optimal fluorescence protocol for studying FBXO10 localization to mitochondria?

For optimal visualization of FBXO10 at mitochondria:

• Cell preparation: Culture cells on glass coverslips coated with poly-L-lysine to improve adherence. Use cells expressing low-to-moderate levels of FBXO10 to avoid artifacts from overexpression .

• Fixation method: Use 4% paraformaldehyde for 15 minutes at room temperature to preserve mitochondrial morphology while maintaining fluorophore activity. Avoid methanol fixation as it can disrupt mitochondrial membrane integrity .

• Permeabilization: Employ 0.1% Triton X-100 for 10 minutes, which allows antibody penetration while preserving mitochondrial architecture .

• Mitochondrial co-staining: Include a mitochondrial marker such as MitoTracker or TOM20 antibody to confirm co-localization. This is critical for distinguishing authentic mitochondrial localization from cytosolic distribution .

• Imaging parameters: Collect Z-stack images at 0.2-0.3 μm intervals using confocal microscopy to accurately capture the three-dimensional distribution of FBXO10 across mitochondrial networks .

• Quantification: Apply flow cytometry of isolated intact mitochondria with approximately 10-20 μg/mL of FITC-conjugated antibody to quantitatively assess mitochondrial association .

How can I assess the impact of geranylgeranylation on FBXO10 mitochondrial localization?

To investigate geranylgeranylation's role in FBXO10 mitochondrial targeting:

• Pharmacological inhibition: Treat cells expressing FBXO10 with specific inhibitors:

  • GGTi-2418 (geranylgeranylation inhibitor)

  • FTi-lonafarnib (farnesylation inhibitor)

  • Lovastatin (mevalonate pathway inhibitor)

  Compare subcellular distribution using confocal microscopy. As demonstrated in previous research, GGTi-2418 and lovastatin, but not FTi-lonafarnib, redistribute FBXO10 away from mitochondria, confirming geranylgeranylation-specific targeting .

• Mutational analysis: Express wild-type FBXO10 alongside FBXO10(C953S) CaaX-motif mutant. The C953S mutation prevents geranylgeranylation and results in delocalization from mitochondria .

• Metabolic labeling: Perform in vivo labeling with geranylgeranyl-azide followed by click chemistry to directly visualize geranylgeranylated FBXO10. This provides direct biochemical evidence of this post-translational modification .

• Quantitative assessment:

  • Flow cytometry of isolated mitochondria to measure FBXO10 association

  • Subcellular fractionation followed by western blotting

  • Live-cell imaging with fluorescently-tagged constructs

These complementary approaches provide robust evidence for geranylgeranylation-dependent targeting of FBXO10 to mitochondria, as evidenced by previous findings .

What experimental approaches can identify authentic FBXO10 substrates at the outer mitochondrial membrane?

Identifying bona fide FBXO10 substrates requires a multi-layered approach:

• Quantitative proteomics workflow:

  • Compare enriched mitochondrial fractions from cells expressing:

    • Wild-type FBXO10

    • CaaX-mutant FBXO10(C953S)

    • Empty vector control

  • Focus on proteins showing reciprocal abundance changes (increased with FBXO10(C953S), decreased with wild-type FBXO10)

  • Filter candidates based on known OMM localization

• Validation of ubiquitylation:

  • Immunoprecipitate candidate substrates and probe for ubiquitin

  • Perform in vitro ubiquitylation assays with reconstituted SCF^FBXO10 complex

  • Use proteasome inhibitors to capture ubiquitylated intermediates

• Direct interaction studies:

  • Co-immunoprecipitation of FBXO10 with candidate substrates

  • Proximity labeling approaches (BioID or APEX) to identify proteins in close proximity to FBXO10 at the OMM

  • Yeast two-hybrid or mammalian two-hybrid to validate direct protein-protein interactions

• Functional validation:

  • Assess protein half-life in FBXO10 knockout versus wild-type cells

  • Examine accumulation of substrates upon proteasome inhibition

  • Test substrate mutants resistant to FBXO10-mediated degradation

This strategy has successfully identified phosphoglycerate mutase 5 (PGAM5) as a validated FBXO10 substrate at the OMM .

How can I investigate the role of HSP90 in FBXO10 mitochondrial targeting?

To examine HSP90's function in FBXO10 mitochondrial localization:

• Pharmacological inhibition:

  • Treat cells with CCT018159 (HSP90 inhibitor) or PES-Cl (HSP70 inhibitor)

  • Assess FBXO10 localization through confocal microscopy, flow cytometry of isolated mitochondria, and subcellular fractionation

  • Previous research demonstrates that inhibiting HSP90, but not HSP70, results in FBXO10 delocalization from mitochondria

• siRNA knockdown approach:

  • Deplete HSP90 and HSP70 using targeted siRNAs

  • Quantify changes in FBXO10 mitochondrial association

  • Research has shown that reduction of HSP90, but not HSP70, causes redistribution of FBXO10 away from mitochondria

• Protein interaction analysis:

  • Immunoprecipitate FBXO10 and probe for HSP90, HSP70, and TOM70

  • Compare binding patterns between wild-type FBXO10 and geranylgeranylation-deficient FBXO10(C953S)

  • Evidence indicates that FBXO10(C953S) maintains binding to HSP90 and HSP70 but fails to interact with TOM70

• Domain mapping:

  • Generate truncated FBXO10 constructs to identify domains required for HSP90 interaction

  • Create HSP90 mutants defective in client protein binding

  • Test these constructs for altered mitochondrial targeting

These approaches collectively establish the essential role of HSP90 in FBXO10 targeting to mitochondria and elucidate the molecular pathway of geranylgeranylation-dependent mitochondrial localization .

How should researchers interpret changes in FBXO10 localization following pharmacological interventions?

When analyzing FBXO10 localization changes after drug treatments:

• Pattern analysis: Distinguish between different subcellular distribution patterns:

  • Punctate mitochondrial pattern (wild-type localization)

  • Homogeneous cytosolic distribution with negatively visualized organelles (delocalization)

  • Mixed patterns indicating partial delocalization

• Quantitative assessment:

  • Flow cytometry of isolated mitochondria provides objective quantification of FBXO10 association with mitochondria

  • Multiple technical replicates (n≥3) are essential for statistical validation

  • Compare mean fluorescence intensity ratios between mitochondrial and cytosolic fractions

• Time-course considerations:

  • Examine acute versus chronic effects of inhibitors

  • Determine the kinetics of FBXO10 redistribution

  • Assess whether effects are reversible upon drug washout

• Mechanistic interpretation:

  • GGTi-2418 effects indicate direct dependence on geranylgeranylation

  • Lovastatin effects suggest broader mevalonate pathway involvement

  • HSP90 inhibitor effects reveal chaperone-dependent targeting

• Functional correlation:

  • Connect localization changes to alterations in mitochondrial function

  • Assess whether mislocalized FBXO10 retains partial function or exhibits dominant-negative effects

  • Determine if substrate degradation correlates with localization changes

Research demonstrates that both inhibition of geranylgeranylation and HSP90 function result in FBXO10 delocalization from mitochondria, supporting a model where these factors cooperatively control mitochondrial targeting .

What considerations are important when analyzing mass spectrometry data for FBXO10-associated proteins?

When interpreting proteomics data for FBXO10-associated proteins:

• Filtering strategies:

  • Apply stringent false discovery rate (FDR) thresholds (≤5%)

  • Set fold-change cutoffs (≥2-fold) for biological significance

  • Filter based on subcellular localization (prioritize OMM-associated proteins)

• Reciprocal abundance patterns:

  • Focus on proteins showing inverse relationship between wild-type FBXO10 and FBXO10(C953S)

  • True substrates typically increase with FBXO10(C953S) and decrease with wild-type FBXO10

  • Non-substrate interactors may show different patterns

• Functional annotation clustering:

  • Analyze enriched biological processes and cellular components

  • Prior research shows enrichment for mitochondria, protein translation, carbohydrate metabolism, oxidative phosphorylation, and mRNA processing

• Network analysis:

  • Build protein interaction networks to identify functional modules

  • Distinguish between direct FBXO10 interactors and downstream effects

  • Integrate with known mitochondrial protein interaction datasets

• Validation prioritization:

  • Prioritize candidates with established OMM localization

  • Consider proteins with known roles in mitochondrial function

  • Evaluate conservation across species to identify fundamental pathways

Research has successfully employed this approach to identify eighteen OMM and/or OMM-associated protein targets of FBXO10 with significant reciprocal abundance changes, including validated substrate PGAM5 .

How can researchers distinguish between direct and indirect effects of FBXO10 on mitochondrial function?

Differentiating direct from indirect effects requires rigorous experimental design:

• Acute versus chronic manipulation:

  • Use inducible expression systems with controlled doxycycline dosage to achieve near-endogenous expression levels

  • Implement rapid protein degradation systems (e.g., auxin-inducible degron) for acute FBXO10 depletion

  • Compare immediate versus delayed phenotypes

• Substrate-specific interventions:

  • Express degradation-resistant substrate mutants

  • Perform rescue experiments with individual FBXO10 substrates in FBXO10-knockout backgrounds

  • Use structure-function analysis to create FBXO10 mutants that selectively lose interaction with specific substrates

• Temporal analysis of molecular events:

  • Establish timeline of:

    • FBXO10 localization changes

    • Substrate accumulation

    • Mitochondrial functional alterations

    • Morphological changes

  • Direct effects should manifest earlier than indirect consequences

• Mitochondrial subcompartment analysis:

  • Distinguish effects on:

    • Outer membrane integrity

    • Intermembrane space proteins

    • Inner membrane potential

    • Matrix function

  • FBXO10 resides in the OMM but not IMM or matrix, so direct effects should predominate at the OMM

• Pathway inhibition approach:

  • Selectively block downstream signaling pathways

  • Determine which mitochondrial phenotypes persist versus resolve

This systematic approach helps establish causality between FBXO10 activity and observed mitochondrial phenotypes, critical for accurate interpretation of experimental results .

What strategies can address inconsistent detection of FBXO10 in mitochondrial fractions?

Inconsistent mitochondrial detection may result from several factors:

• Optimization of mitochondrial isolation:

  • Use differential centrifugation with sucrose gradient purification

  • Verify mitochondrial enrichment using markers for different compartments (TOM20 for OMM, COX IV for IMM)

  • Ensure minimal contamination from other organelles (ER, peroxisomes)

• Antibody selection considerations:

  • Test multiple antibody clones targeting different FBXO10 epitopes

  • Optimize antibody concentration (start with 10-20 μg/mL for FITC-conjugated antibodies)

  • Validate with positive controls (cells overexpressing FBXO10)

• Fixation and permeabilization optimization:

  • Compare different fixatives (paraformaldehyde, glutaraldehyde)

  • Test various permeabilization agents (Triton X-100, digitonin, saponin)

  • Digitonin at low concentrations (0.01-0.05%) can selectively permeabilize the plasma membrane while preserving mitochondrial membranes

• Expression level considerations:

  • Endogenous FBXO10 levels may be near detection limits

  • Use doxycycline-inducible systems for controlled expression

  • Consider signal amplification methods for low-abundance detection

• Physiological state awareness:

  • FBXO10 localization may vary with cell cycle, stress conditions, or metabolic state

  • Standardize culture conditions and time points for consistent results

  • Document cell confluence and passage number for reproducibility

These approaches collectively address technical variables that can impact FBXO10 detection in mitochondrial fractions .

How can researchers overcome challenges in quantifying FBXO10-substrate interactions?

Quantifying FBXO10-substrate interactions presents several challenges:

• Transient interaction capture:

  • Use crosslinking approaches (formaldehyde, DSP, photo-crosslinkers)

  • Implement proteasome inhibitors (MG132, bortezomib) to stabilize ubiquitylated intermediates

  • Utilize UBA domain fusion proteins to trap ubiquitylated substrates

• Reduction of background signal:

  • Employ tandem affinity purification strategies

  • Include stringent washing steps with detergents (0.1% SDS, 1% Triton X-100)

  • Use denaturing conditions followed by renaturation for specific isolation of covalently modified substrates

• Quantitative co-immunoprecipitation:

  • Include internal standards for normalization

  • Implement SILAC or TMT labeling for mass spectrometry-based quantification

  • Use fluorescently-tagged proteins with calibration curves for stoichiometry determination

• In situ interaction assessment:

  • Apply proximity ligation assay (PLA) for visualizing protein interactions in intact cells

  • Implement FRET or BRET approaches for live-cell interaction monitoring

  • Correlation of signal with functional outcomes provides validation

• Competitive binding analysis:

  • Use peptide competition assays to map interaction domains

  • Implement heterologous competition experiments to determine relative binding affinities

  • Develop in vitro reconstitution systems with purified components

These methodological approaches enhance detection specificity and quantitative accuracy for FBXO10-substrate interactions, critical for validating authentic substrates .

What emerging technologies could advance FBXO10 research at the mitochondrial interface?

Several cutting-edge approaches hold promise for FBXO10 research:

• Spatially-resolved proteomics:

  • Proximity-dependent biotinylation (BioID, TurboID) targeted to the OMM

  • APEX2-based labeling for temporal resolution of the FBXO10 interactome

  • Hyperplexed imaging mass cytometry for spatial organization of FBXO10 complexes

• Advanced live-cell imaging:

  • Super-resolution microscopy (STED, PALM, STORM) to visualize FBXO10 nanoscale organization

  • Light-sheet microscopy for long-term tracking of FBXO10 dynamics with minimal phototoxicity

  • Optogenetic tools for spatiotemporal control of FBXO10 activity

• Single-cell approaches:

  • Single-cell proteomics to capture cell-to-cell variability in FBXO10 function

  • Correlative light and electron microscopy (CLEM) to link FBXO10 localization with ultrastructural changes

  • Microfluidic-based single-cell analysis of mitochondrial function

• Structural biology integration:

  • Cryo-electron microscopy of FBXO10-containing complexes at the OMM

  • Integrative structural modeling combining crosslinking mass spectrometry, SAXS, and computational approaches

  • In-cell NMR to study FBXO10 conformational changes upon substrate binding

• Genome engineering innovations:

  • CRISPR-mediated endogenous tagging for physiological studies

  • Base editing to introduce specific mutations in FBXO10 or its substrates

  • Screening approaches to identify synthetic lethal interactions with FBXO10 dysfunction

These technologies will provide unprecedented insights into how geranylgeranylated-SCF^FBXO10 regulates selective outer mitochondrial membrane proteostasis and function .

How might FBXO10 research connect to broader questions in mitochondrial quality control?

FBXO10 research intersects with several fundamental areas of mitochondrial biology:

• Integration with other quality control systems:

  • Relationship between FBXO10-mediated protein turnover and mitophagy

  • Coordination with mitochondrial unfolded protein response

  • Interplay with proteases like PARL, OMA1, and YME1L that regulate mitochondrial dynamics

• Metabolic regulation:

  • Impact of FBXO10 substrates on oxidative phosphorylation

  • Connection between protein turnover and carbohydrate metabolism

  • Role in mitochondrial adaptation to nutrient availability

• Disease relevance:

  • Potential dysregulation in neurodegenerative disorders characterized by mitochondrial dysfunction

  • Connection to cancer metabolism through altered mitochondrial function

  • Implications for aging-related mitochondrial decline

• Evolutionary perspectives:

  • Conservation of FBXO10-mediated regulation across species

  • Adaptation of this system in organisms with different metabolic requirements

  • Co-evolution with mitochondrial architecture and function

• Therapeutic potential:

  • Targeting the geranylgeranylation pathway (connection to statin effects)

  • Modulating HSP90-dependent mitochondrial protein targeting

  • Selective manipulation of FBXO10 substrates for mitochondrial protection

The emerging understanding of FBXO10's role provides a molecular framework connecting protein quality control, post-translational modifications, and mitochondrial homeostasis, with implications for both fundamental biology and disease intervention strategies .