FBXO15 Antibody

What is FBXO15 and what cellular functions does it regulate?

FBXO15 (F-box protein 15) is a member of the F-box protein family that functions as a substrate recognition component within the SCF (Skp1-Cullin1-F-box) ubiquitin E3 ligase complex. F-box proteins are critical for the ubiquitin-mediated degradation of cellular regulatory proteins and are characterized by an approximately 40 amino acid F-box motif . FBXO15 plays significant roles in several cellular processes through its ability to target specific proteins for ubiquitination and subsequent proteasomal degradation.

Research has identified multiple substrates of FBXO15, including:

• CLS1 (Cardiolipin Synthase 1), where FBXO15-mediated degradation impacts mitochondrial integrity and function

• P-glycoprotein/ABCB1, where FBXO15 regulation affects multidrug resistance in cancer cells

Additionally, FBXO15 is expressed predominantly in undifferentiated embryonic stem cells and has been identified as a target of the pluripotency transcription factor Oct3/4, suggesting a potential role in stem cell biology .

What are the validated applications for FBXO15 antibodies in research?

FBXO15 antibodies have been validated for multiple research applications, with specific methodological considerations for each:

For all applications, it is recommended to titrate the antibody concentration to obtain optimal results for each specific experimental system and sample type .

How should researchers validate FBXO15 antibody specificity for their experimental systems?

Proper validation of FBXO15 antibody specificity is crucial for reliable experimental results. Implement the following methodological approaches:

• Positive and negative controls:

  • Use cell lines or tissues with confirmed FBXO15 expression (e.g., A2780 cells, mouse ovary tissue) as positive controls

  • Include FBXO15 knockout or knockdown samples (using FBXO15-shRNA or siRNA) as negative controls

• Molecular weight verification:

  • Confirm detection at the expected molecular weight (~55 kDa observed; 49 kDa calculated)

  • Check for any non-specific bands that might interfere with interpretation

• Cross-reactivity assessment:

  • Test antibody reactivity across species if working with non-human models (validated for human and mouse samples)

  • Perform blocking peptide competition assays to confirm epitope specificity

• Alternative antibody comparison:

  • Compare results using antibodies targeting different epitopes of FBXO15

  • Correlate protein detection with mRNA expression data

• Functional validation:

  • Verify that FBXO15 knockdown corresponds with expected changes in substrate protein levels (e.g., increased P-glycoprotein or CLS1 levels)

How can researchers design experiments to study FBXO15-mediated protein degradation pathways?

When investigating FBXO15's role in protein degradation pathways, consider these methodological approaches:

• Ubiquitination assays:

  • Transfect cells with FLAG-tagged ubiquitin and/or HA-tagged FBXO15 constructs

  • Treat cells with proteasome inhibitors (e.g., MG132) 4-6 hours before harvesting to accumulate ubiquitinated proteins

  • Immunoprecipitate the substrate protein of interest and immunoblot for ubiquitin to detect ubiquitination

• Protein stability assays:

  • Perform cycloheximide chase experiments to measure substrate protein half-life (t1/2)

  • Compare protein degradation kinetics between wild-type cells and those with FBXO15 overexpression or knockdown

  • Include proteasome inhibitors (MG132) and lysosomal inhibitors (leupeptin) to distinguish between degradation pathways

• Substrate identification:

  • Utilize immunoprecipitation followed by mass spectrometry to identify novel FBXO15-interacting proteins

  • Verify interactions through reciprocal co-immunoprecipitation experiments

  • Create lysine-to-arginine mutants of potential substrate proteins to identify ubiquitination sites (e.g., K174R in CLS1)

• SCF complex analysis:

  • Investigate the association of FBXO15 with other SCF components (Skp1, Cullin1)

  • Use truncated mutants to identify domains required for substrate recognition and complex formation

• Functional consequences:

  • Assess biological outcomes of FBXO15-mediated degradation (e.g., mitochondrial morphology changes, membrane potential alterations, ATP production)

  • Compare phenotypes between wild-type cells and those with altered FBXO15 expression

What methodological approaches can address heterogeneous FBXO15 expression across different cell types?

Researchers encountering variable FBXO15 expression across experimental models should consider these methodological strategies:

• Comprehensive expression profiling:

  • Perform systematic analysis of FBXO15 protein levels across relevant cell types using Western blot

  • Correlate with mRNA expression data from RT-qPCR

  • Consider single-cell analysis approaches (e.g., flow cytometry, CyTOF) to assess cell-to-cell variability

• Regulatory mechanism investigation:

  • Analyze FBXO15 promoter activation in different cell types, particularly focusing on Oct3/4 binding sites

  • Use ChIP assays to examine transcription factor binding at the FBXO15 promoter

  • Investigate post-transcriptional regulation through miRNA profiling

• Alternative isoform detection:

  • Design primers to detect potential alternative FBXO15 splice variants

  • Use antibodies recognizing different epitopes to identify isoform-specific expression patterns

  • Perform 5' RACE to identify transcription initiation sites in different cell types

• Controlled expression systems:

  • Utilize inducible expression systems to achieve consistent FBXO15 levels across cell types

  • Consider viral transduction methods for cells that are difficult to transfect

  • Generate stable cell lines with uniform FBXO15 expression

• Tissue-specific functionality:

  • Compare substrate targeting efficiency across cell types with different endogenous FBXO15 levels

  • Investigate cell-specific co-factors that might influence FBXO15 activity

What optimization strategies improve immunohistochemical detection of FBXO15 in tissue samples?

For optimal FBXO15 detection in tissue sections, researchers should consider these methodological refinements:

• Antigen retrieval optimization:

  • Primary recommendation: Use TE buffer pH 9.0 for heat-induced epitope retrieval

  • Alternative option: Citrate buffer pH 6.0 if TE buffer yields suboptimal results

  • Systematically test retrieval times (10-30 minutes) to find optimal conditions

• Antibody concentration titration:

  • Begin with the recommended 1:200-1:800 dilution range

  • Perform a dilution series to determine optimal signal-to-noise ratio for specific tissue types

  • Consider tissue-specific optimization, as FBXO15 has shown differential detection in various tissues

• Detection system selection:

  • For tissues with low FBXO15 expression, use amplification-based detection systems

  • For co-localization studies, select fluorescent secondary antibodies compatible with other markers

  • Include appropriate controls using tissues with known FBXO15 expression (e.g., ovarian cancer tissue)

• Tissue preparation considerations:

  • Compare fresh-frozen versus formalin-fixed paraffin-embedded (FFPE) samples

  • Minimize fixation time to prevent excessive protein crosslinking

  • Consider performing section thickness optimization (4-8 μm)

• Background reduction strategies:

  • Implement proper blocking steps with serum from the same species as the secondary antibody

  • Include endogenous peroxidase quenching step for chromogenic detection

  • Add avidin/biotin blocking for biotin-based detection systems

How should researchers troubleshoot discrepancies between FBXO15 antibody detection methods?

When encountering inconsistencies between different detection methods for FBXO15, implement these methodological approaches:

• Epitope accessibility assessment:

  • Different applications (WB, IHC, ICC) expose different epitopes

  • Under reducing conditions (WB), epitopes may be more accessible than in fixed samples (IHC/ICC)

  • Test multiple antibodies targeting different regions of FBXO15

• Sample preparation comparison:

  • For Western blot: Compare different lysis buffers (RIPA vs. NP-40) and extraction methods

  • For IHC/ICC: Evaluate different fixatives (paraformaldehyde, methanol, acetone)

  • For flow cytometry: Optimize permeabilization conditions for intracellular staining

• Sensitivity threshold determination:

  • Establish detection limits for each method using serial dilutions of recombinant FBXO15

  • Consider signal amplification methods for samples with low expression

  • Use more sensitive detection methods (e.g., chemiluminescence for WB) for low-abundance samples

• Protein modification interference:

  • Investigate if post-translational modifications affect antibody recognition

  • Include phosphatase or deubiquitinase treatments to remove modifications that might mask epitopes

  • Consider protein complex formation that might sequester epitopes

• Methodological workflow standardization:

  • Implement consistent protocols across experiments

  • Document all variables that could affect detection (incubation times, temperatures, buffer compositions)

  • Maintain detailed records of antibody lot numbers and storage conditions

How can researchers distinguish between FBXO15 isoforms in experimental samples?

FBXO15 has multiple isoforms, and proper identification requires systematic analytical approaches:

• Molecular weight profiling:

  • The calculated molecular weight of FBXO15 is 49 kDa, but it typically appears at approximately 55 kDa on SDS-PAGE

  • Different isoforms may appear as distinct bands (e.g., isoform 2 encompasses amino acids 298-434 of the full-length protein)

  • Use high-resolution gel systems (e.g., gradient gels) to resolve closely-migrating isoforms

• Isoform-specific antibody selection:

  • Choose antibodies targeting regions that differ between isoforms

  • Consider using antibodies raised against specific isoforms when available

  • Validate antibody specificity with recombinant protein standards of each isoform

• Transcript analysis correlation:

  • Perform RT-PCR with isoform-specific primers

  • Use 5' RACE to identify alternative transcription start sites

  • Correlate mRNA isoform expression with protein band patterns

• Genetic modification approaches:

  • Design isoform-specific siRNAs or shRNAs to selectively deplete individual variants

  • Express tagged versions of specific isoforms as positive controls

  • Use CRISPR/Cas9 to target isoform-specific exons

• Mass spectrometry verification:

  • Perform immunoprecipitation followed by mass spectrometry

  • Identify isoform-specific peptides to confirm the presence of particular variants

  • Compare experimental spectra with theoretical peptide maps of known isoforms

What experimental controls are essential when studying FBXO15-mediated ubiquitination processes?

Rigorous investigation of FBXO15's role in ubiquitination requires these control experiments:

• Substrate specificity controls:

  • Include positive control substrates with confirmed FBXO15-dependent ubiquitination (e.g., CLS1, P-glycoprotein)

  • Test proteins not targeted by FBXO15 as negative controls

  • Use lysine-to-arginine mutants of target proteins to confirm specific ubiquitination sites

• E3 ligase component controls:

  • Compare FBXO15 with other F-box proteins (e.g., conduct parallel experiments with FBXL or FBXW family members)

  • Include dominant-negative SCF complex components

  • Test FBXO15 mutants lacking the F-box domain to verify SCF complex formation requirement

• Proteasome inhibition controls:

  • Include samples with proteasome inhibitors (e.g., MG132) to accumulate ubiquitinated proteins

  • Compare with lysosomal inhibitors (e.g., leupeptin) to distinguish between degradation pathways

  • Use graded concentrations and time courses of inhibitor treatment

• Genetic manipulation controls:

  • Include FBXO15 knockdown/knockout controls using validated siRNA or shRNA constructs

  • Perform rescue experiments with siRNA-resistant FBXO15 constructs

  • Use wild-type cells alongside genetically modified lines

• Ubiquitination assay controls:

  • Include samples without tagged ubiquitin as background controls

  • Use deubiquitinating enzyme inhibitors to preserve ubiquitin modifications

  • Perform experiments with ubiquitin mutants (e.g., K48R, K63R) to identify linkage types

How can FBXO15 antibodies advance research on mitochondrial dysfunction mechanisms?

FBXO15 has been implicated in mitochondrial function regulation through its targeting of CLS1, making it valuable for mitochondrial research:

• Cardiolipin synthesis pathway investigation:

  • Use FBXO15 antibodies to monitor endogenous levels in models of mitochondrial disease

  • Correlate FBXO15 expression with cardiolipin content using mass spectrometry

  • Study FBXO15-CLS1 interaction in response to mitochondrial stressors

• Mitochondrial morphology assessment:

  • Combine FBXO15 immunostaining with mitochondrial markers to correlate expression with morphological changes

  • Implement high-content imaging to quantify mitochondrial parameters (size, density, network connectivity)

  • Track temporal dynamics of FBXO15 expression and mitochondrial remodeling

• Mitochondrial membrane potential analysis:

  • Use JC-1 or DiOC2(3) staining in conjunction with FBXO15 expression analysis

  • Perform flow cytometry-based studies to correlate FBXO15 levels with membrane potential at single-cell resolution

  • Conduct live-cell imaging of membrane potential in cells with modulated FBXO15 expression

• ATP production measurement:

  • Quantify ATP levels in relation to FBXO15 expression

  • Implement luciferase-based assays to monitor real-time ATP dynamics

  • Correlate FBXO15-mediated effects with respiratory chain complex activities

• Disease model applications:

  • Examine FBXO15 levels in models of pneumonia-associated acute lung injury

  • Investigate FBXO15-PINK1 pathway interactions in Parkinson's disease models

  • Study therapeutic targeting of FBXO15 to preserve mitochondrial integrity

What methodological approaches can determine FBXO15 substrate specificity across different tissues and disease states?

Investigating tissue-specific and disease-related functions of FBXO15 requires these systematic approaches:

• Comparative interactome analysis:

  • Perform immunoprecipitation of FBXO15 followed by mass spectrometry across different tissues

  • Compare protein interaction networks between normal and disease states

  • Validate tissue-specific interactions with co-immunoprecipitation and proximity ligation assays

• Substrate competition assays:

  • Develop in vitro ubiquitination assays with purified components

  • Test multiple potential substrates simultaneously to assess preferential targeting

  • Investigate how tissue-specific factors might influence substrate selection

• Domain mapping experiments:

  • Generate truncated or point-mutated FBXO15 constructs

  • Identify regions responsible for tissue-specific substrate recognition

  • Perform structural analysis of FBXO15-substrate complexes

• Phosphorylation-dependent regulation:

  • Investigate how PINK1 kinase activity affects FBXO15-substrate interactions

  • Examine phosphorylation sites on FBXO15 and potential substrates

  • Use phosphomimetic and phospho-deficient mutants to determine functional consequences

• Disease-specific expression profiling:

  • Compare FBXO15 levels across tissues in disease models (cancer, neurodegenerative disorders)

  • Correlate expression with disease progression markers

  • Develop tissue-specific conditional knockout models to assess function in vivo