FBXO7 Antibody

Basic Research Questions

• What is FBXO7 and why are antibodies against it important in research?

  FBXO7 (F-box protein 7) functions as a substrate recognition component of SCF (SKP1-CUL1-F-box protein) E3 ubiquitin-protein ligase complex that mediates ubiquitination and subsequent proteasomal degradation of target proteins. The human canonical protein consists of 522 amino acid residues with a mass of approximately 58.5 kDa . FBXO7 plays critical roles in cell cycle regulation, cell proliferation, and maintenance of chromosome stability. It has gained significant research interest due to its implications in Parkinson's disease (identified as PARK15), mitochondrial function, inflammation, and various cancers . Antibodies against FBXO7 are essential tools for studying its expression, localization, interactions, and functions in these pathological contexts.

• What are the primary applications of FBXO7 antibodies in research?

  FBXO7 antibodies are employed in multiple experimental applications, with Western blotting being the most widely used method. The table below summarizes key applications and their relative frequency of use:

  Application	Frequency of Use	Notes
  Western Blot (WB)	Very Common	Detection of FBXO7 protein expression levels
  Immunohistochemistry (IHC)	Common	Tissue localization studies
  Immunoprecipitation (IP)	Common	Protein-protein interaction studies
  ELISA	Less Common	Quantitative protein detection
  Immunocytochemistry/Immunofluorescence (ICC/IF)	Less Common	Subcellular localization

  When selecting an antibody, researchers should verify that it has been validated for their specific application .

• What are the typical molecular weight and localization patterns observed when using FBXO7 antibodies?

  When using FBXO7 antibodies for Western blotting, researchers should expect to detect bands ranging from 59-75 kDa, with the canonical form at approximately 58.5 kDa . The variation in observed molecular weight can result from post-translational modifications or different isoforms (up to 3 isoforms have been reported) .

  Regarding subcellular localization, FBXO7 has been detected in multiple cellular compartments:

  • Nucleus (primary localization)

  • Mitochondria (particularly important for its mitophagy-related functions)

  • Cytoplasm

  Immunofluorescence studies should account for this multi-compartmental distribution, and appropriate co-localization markers should be employed to validate specific subcellular pools of FBXO7 .

• How should FBXO7 antibodies be stored and handled to maintain optimal activity?

  For optimal performance, FBXO7 antibodies should be handled according to these guidelines:

  • Storage temperature: -20°C for long-term storage

  • Short-term storage: 2-8°C for up to one week

  • Buffer conditions: PBS with 0.02% sodium azide and 50% glycerol at pH 7.3

  • Aliquoting: For antibodies intended for frequent use, divide into small aliquots to prevent repeated freeze-thaw cycles

  • Working dilutions: Typically 1:500-1:4000 for Western blotting and 1:50-1:500 for immunohistochemistry, but optimal dilutions should be determined empirically for each specific application and sample type

  Note that some formulations may contain additional components like BSA (0.1%) for stabilization .

• Which cell lines and tissues are most appropriate for validating FBXO7 antibodies?

  Several cell lines and tissues have been documented as appropriate positive controls for FBXO7 antibody validation:

  Cell Lines	Tissues
  HeLa	Human stomach tissue
  MCF-7	Human intrahepatic cholangiocarcinoma
  Raji	Human brain tissue (particularly for neurodegenerative research)
  HEK293T	Lymphoid tissues (high expression level)

  Notably, FBXO7 is highly expressed in hematopoietic and lymphoid lineages, making these excellent positive controls. For negative controls, FBXO7 knockout cell lines or CRISPR-edited lines with FBXO7 deletion should be used when possible .

Advanced Research Questions

• How can FBXO7 antibodies be utilized to investigate its role in the PINK1/Parkin mitophagy pathway?

  Investigating FBXO7's role in the PINK1/Parkin pathway requires careful experimental design and selection of appropriate antibodies. Recent research presents contradictory findings regarding FBXO7's exact function in mitophagy:

  • Some studies suggest FBXO7 mediates PINK1 ubiquitylation and degradation, indicating a negative regulatory role

  • Contrasting research claims FBXO7 is dispensable for PINK1/Parkin-mediated mitophagy in various cell culture systems

  Methodological approach:

  1. Use FBXO7 antibodies in combination with anti-PINK1 antibodies for co-immunoprecipitation studies to assess direct interaction

  2. Perform time-course Western blotting following mitochondrial depolarization (using CCCP or antimycin/oligomycin) to track PINK1 stabilization and FBXO7 recruitment

  3. Employ super-resolution microscopy with FBXO7 antibodies and mitochondrial markers to visualize recruitment dynamics

  4. Compare results between wild-type, FBXO7-deficient, and PINK1-deficient cellular systems

  5. Validate findings across multiple cell types, including neuronal models more relevant to Parkinson's disease

  When interpreting results, researchers should consider that FBXO7's role may be context-dependent or cell-type specific .

• What considerations are important when using FBXO7 antibodies to study its E3 ubiquitin ligase activity and substrates?

  Studying FBXO7's E3 ligase activity requires specialized approaches:

  1. Target substrate selection: FBXO7 has multiple confirmed substrates, including:

     • BIRC2 (cellular inhibitor of apoptosis)

     • DLGAP5 (cell cycle regulator)

     • PINK1 (mitochondrial kinase)

     • UXT isoform 2 (affecting NF-kB signaling)

     • TRAF2 (TNF receptor-associated factor)

     • PSMA2 (proteasomal subunit)

     • SIRT7 (sirtuin 7)

     • MiD49/51 (mitochondrial dynamics proteins)

     • PFKP (phosphofructokinase, platelet type - glycolysis regulator)

  2. Ubiquitination patterns: FBXO7 can mediate different types of ubiquitin linkages:

     • K48-linked polyubiquitin (targeting for degradation) - e.g., for SIRT7

     • K63-linked polyubiquitin (non-degradative signaling) - e.g., for PSMA2 and Rbfox2

  3. Experimental design for ubiquitination assays:

     • Use in vitro ubiquitination assays with purified components (E1, E2, FBXO7 in SCF complex, substrate, ubiquitin)

     • Perform immunoprecipitation of substrate followed by ubiquitin immunoblotting

     • Compare wild-type FBXO7 with F-box domain mutants that disrupt SCF complex formation

     • Consider using ubiquitin linkage-specific antibodies to determine chain type

     • Include proteasome inhibitors (e.g., MG132) to prevent degradation of ubiquitinated substrates

  4. Complementary approaches:

     • Size exclusion chromatography to study complex formation between FBXO7, SKP1, CUL1 and substrates

     • FBXO7 domain mapping to identify substrate-binding regions

     • Mass spectrometry to identify ubiquitination sites on substrates

• How should researchers address contradictory findings regarding FBXO7's role in cancer progression?

  The literature contains contradictory reports on FBXO7's role in cancer, presenting both tumor-promoting and tumor-suppressive functions. When designing experiments to address these contradictions, consider:

  1. Cancer type specificity:

     • FBXO7 shows high expression and dependency in hematopoietic and lymphoid malignancies

     • Different roles have been reported in lung, colorectal, and glioblastoma cancers

  2. Methodological approaches to resolve contradictions:

     • Use tissue microarrays with FBXO7 antibodies to evaluate expression across multiple cancer types

     • Correlate FBXO7 expression with patient outcomes and molecular subtypes

     • Perform both gain-of-function and loss-of-function studies in the same cancer model

     • Analyze substrate specificity in different cancer contexts

     • Investigate specific post-translational modifications of FBXO7 in different cancers

  3. Important recent findings:

     • In glioblastoma, FBXO7 confers mesenchymal properties and chemoresistance by controlling Rbfox2-mediated alternative splicing

     • FBXO7 ubiquitinates Rbfox2 through K63-linked chains following arginine dimethylation by PRMT5

     • High FBXO7 protein levels correlate with mesenchymal markers (CD44 & Vimentin) in glioblastoma specimens

  Notably, FBXO7 protein levels, rather than mRNA levels, correlate with mesenchymal phenotypes in glioblastoma, suggesting post-transcriptional regulation is critical for its function in cancer .

• What techniques can be used to investigate FBXO7's role in regulating T cell metabolism through PFKP and Cdk6?

  Recent research has identified FBXO7's role in regulating T cell metabolism through interactions with PFKP (phosphofructokinase, platelet type) and Cdk6. To study this function:

  1. Experimental approaches for FBXO7-Cdk6-PFKP interaction:

     • Co-immunoprecipitation with FBXO7 antibodies followed by blotting for Cdk6 and PFKP

     • Proximity ligation assays to visualize protein interactions in situ

     • In vitro kinase assays to measure Cdk6 activity with/without FBXO7

  2. Techniques for studying PFKP regulation:

     • Size exclusion chromatography to analyze PFKP tetramer formation (active) versus dimer/monomer (less active) states

     • Phosphorylation-specific antibodies to detect Cdk6-mediated phosphorylation of PFKP

     • Enzymatic assays to measure PFKP activity in FBXO7-deficient versus control cells

  3. Metabolic analyses:

     • Extracellular flux analysis (Seahorse) to measure glycolytic rates in T cells

     • Stable isotope tracing to track glucose metabolism through glycolysis

     • Metabolomics to assess broader impact on purine/pyrimidine synthesis and arginine metabolism

  4. Functional T cell assays:

     • Activation assays using CD3/CD28 stimulation in FBXO7-deficient T cells

     • Viability measurements under different glucose concentrations

     • Proliferation assays correlated with FBXO7 and Cdk6 expression levels

  Key finding: FBXO7 deficiency reduces Cdk6 activity and increases glycolytic flux in T cells, contrary to what might be expected given PFKP's role as a glycolytic enzyme. This highlights the complex regulatory mechanisms involved .

• How can researchers evaluate FBXO7 antibody specificity and validate knockout/knockdown models?

  Ensuring antibody specificity is critical for FBXO7 research, especially given its multiple isoforms and related F-box proteins:

  1. Knockout/knockdown validation strategies:

     • Western blotting on wild-type versus FBXO7 knockout/knockdown samples using multiple FBXO7 antibodies targeting different epitopes

     • qRT-PCR to confirm mRNA reduction (for knockdown) or absence (for knockout)

     • Genomic PCR to confirm CRISPR-mediated gene editing

     • Rescue experiments with FBXO7 cDNA to restore phenotypes

  2. Antibody specificity verification:

     • Pre-adsorption tests using blocking peptides specific to the antibody epitope

     • Cross-reactivity assessment with other F-box proteins

     • Comparison of staining patterns across multiple FBXO7 antibodies

     • Mass spectrometry validation of immunoprecipitated proteins

  3. Recommended controls for FBXO7 studies:

     • Include at least one FBXO7 knockout/knockdown sample in each experiment

     • For immunoprecipitation, include IgG-only controls

     • When possible, include multiple cell types with different FBXO7 expression levels

     • For disease-related studies, test antibodies on patient-derived samples with known FBXO7 mutations

  Recent research has utilized these validation approaches, particularly in establishing the specificity of FBXO7 knockout clones for mitophagy studies .

• What approaches are recommended for studying FBXO7 mutations associated with Parkinson's disease?

  FBXO7 mutations (PARK15) cause a form of atypical parkinsonism. To investigate these mutations:

  1. Structural and functional analysis approaches:

     • Focus on key FBXO7 domains affected by mutations, particularly the dimerization domain (FP domain) and F-box domain

     • Use molecular docking and 3D structure modeling to predict effects of mutations

     • Screen for small molecule inhibitors that may modulate FBXO7 activity, like BC1464 which disrupts FBXO7-PINK1 interaction

  2. Cellular models:

     • Generate cell lines expressing FBXO7 patient mutations (e.g., L250P)

     • Use patient-derived fibroblasts or iPSC-derived neurons

     • Measure proteasome activity and subunit levels in mutant versus wild-type cells

     • Assess mitochondrial function parameters (membrane potential, ROS production)

     • Quantify mitophagy rates using established reporters

  3. Key reported findings:

     • The L250P mutation in the dimerization domain selectively disrupts FBXO7-PI31 interaction

     • Patient fibroblasts with this mutation show reduced proteasome activity

     • PI31 interacts with mitochondrial fission adaptor proteins (MiD49/51)

     • FBXO7 mutations impair SCF-FBXO7 ligase activity toward specific substrates

     • Patient cells show higher ROS levels and reduced viability under stress

  4. Therapeutic approach targeting:

     • Chemical inhibition of FBXO7 reduces inflammation and confers neuroprotection by stabilizing PINK1

     • The compound BC1464 targets the FP domain of FBXO7 and disrupts FBXO7-PINK1 interaction