FBXO2 Antibody

What is FBXO2 and what are its primary cellular functions?

FBXO2, also known as FBG1 or Fbs1, is a member of the F-box protein family that functions as a substrate recognition component of the SCF (SKP1-CUL1-F-box protein) E3 ubiquitin-protein ligase complex . This complex mediates the ubiquitination and subsequent proteasomal degradation of target proteins. FBXO2 specifically recognizes and binds to high-mannose-type asparagine-linked carbohydrate chains (N-glycans) on glycoproteins .

FBXO2 plays a crucial role in the endoplasmic reticulum-associated degradation (ERAD) pathway for misfolded lumenal proteins. It accomplishes this by recognizing and binding sugar chains on unfolded glycoproteins that are retrotranslocated into the cytosol, promoting their ubiquitination and subsequent degradation . Additionally, FBXO2 prevents the formation of cytosolic aggregates of unfolded glycoproteins that have escaped into the cytosol .

Recent research has expanded our understanding of FBXO2's functions, revealing its involvement in xenophagy (antibacterial selective autophagy) through the recognition of GlcNAc side chains on bacterial pathogens like group A Streptococcus .

How is FBXO2 expression regulated in different tissues and disease states?

In colorectal cancer (CRC), FBXO2 expression has been detected in 62.6% of tissue samples, suggesting its relevance in this malignancy . The expression of FBXO2 in cancer tissues appears to have clinical significance, as emerging experimental and clinical data indicate that F-box proteins, including FBXO2, can function as either tumor suppressors or oncoproteins depending on the cellular context .

In neurodegenerative disorders, particularly Alzheimer's disease, FBXO2 has been shown to regulate amyloid precursor protein (APP) levels and processing in the brain, which may play a role in modulating disease pathogenesis .

Which experimental models are most suitable for studying FBXO2 function?

Based on the research literature, several experimental models have proven effective for studying FBXO2 function:

• Cell culture systems: Human cell lines like U-87 MG (human glioblastoma-astrocytoma epithelial cells) have been successfully used for studying FBXO2 expression and function .

• Primary neuron cultures: Primary cultures of hippocampal neurons prepared from mice at postnatal day 3 have been instrumental in studying FBXO2's role in neuronal function .

• FBXO2 knockout models: FBXO2-knockout mice and FBXO2-knockout HeLa cells (using CRISPR/Cas9 genome editing) have been valuable for investigating the physiological roles of FBXO2 in various contexts .

• Bacterial infection models: Systems using group A Streptococcus (GAS) infection in mammalian cells have revealed FBXO2's role in xenophagy .

When selecting an experimental model, researchers should consider the specific aspect of FBXO2 function they wish to investigate, as different models may highlight different functional roles of this multifaceted protein.

How can FBXO2 antibodies be utilized to study xenophagy mechanisms?

FBXO2 antibodies serve as valuable tools for investigating xenophagy mechanisms, particularly in the context of bacterial infections. Recent research has demonstrated that FBXO2 plays a critical role in recognizing GlcNAc side chains on the surface of bacterial pathogens like group A Streptococcus (GAS), thereby promoting ubiquitin-mediated xenophagy .

For studying this mechanism:

• Subcellular localization studies: FBXO2 antibodies can be used in immunofluorescence microscopy to track the recruitment of FBXO2 to intracellular bacteria. Research has shown that EmGFP-FBXO2 is recruited to intracellular GAS in more than 50% of infected cells .

• Co-localization analysis: FBXO2 antibodies can be used alongside LC3 (an autophagy marker) antibodies to determine the temporal relationship between FBXO2 recruitment and autophagosome formation. Studies have revealed that FBXO2 is recruited to GAS more frequently than LC3 at early time points (2 and 4 hours post-infection), suggesting FBXO2 acts upstream of LC3 in the xenophagy pathway .

• Functional studies in knockout systems: By comparing ubiquitination patterns in wild-type versus FBXO2-knockout cells during bacterial infection, researchers can assess the contribution of FBXO2 to bacteria-targeted ubiquitination. FBXO2-knockout has been shown to reduce ubiquitin, p62, and LC3 recruitment to intracellular GAS .

• Analysis of bacterial survival: FBXO2 antibodies can help correlate FBXO2 expression levels with bacterial clearance efficiency. Research has demonstrated that GAS survival significantly increases in FBXO2-knockout cells compared to wild-type cells .

When designing xenophagy experiments, it's important to include appropriate controls, such as comparing responses to wild-type bacteria versus mutants lacking specific surface structures (e.g., GAS lacking GlcNAc residues through gacI deletion) .

What is the prognostic significance of FBXO2 in colorectal cancer and how can antibodies help assess this?

FBXO2 has emerged as a potential prognostic marker in colorectal cancer (CRC). Research has shown that FBXO2 expression can be detected in CRC tissues using immunohistochemistry (IHC) with specific antibodies .

The prognostic significance of FBXO2 in CRC can be assessed through:

• Expression correlation with clinical parameters: FBXO2 antibodies can be used in IHC to evaluate expression patterns in patient samples, which can then be correlated with clinicopathological factors using statistical methods like Chi-squared tests .

• Survival analysis: By categorizing patients based on FBXO2 expression levels (positive vs. negative), researchers can generate Kaplan-Meier survival curves and assess group differences using the Log-rank test. Cox proportional hazards models can further assess the correlation of FBXO2 expression with survival outcomes .

• Multimarker analysis: FBXO2 antibodies can be used alongside other markers like Ki67 (proliferation marker) and N-cadherin (EMT marker) to develop more comprehensive prognostic panels .

When conducting prognostic studies with FBXO2 antibodies, researchers should:

• Use validated antibodies with demonstrated specificity

• Include appropriate positive and negative controls

• Implement standardized scoring systems for IHC

• Perform multivariate analysis to account for potential confounding factors

Proper statistical analysis is crucial, ideally using software like SPSS with a predetermined significance threshold (typically p<0.05) .

How does FBXO2 contribute to neurodegenerative disease pathology?

FBXO2 plays significant roles in neurodegenerative disease pathology, particularly in Alzheimer's disease (AD), and FBXO2 antibodies are essential tools for investigating these mechanisms:

• Regulation of amyloid precursor protein (APP): FBXO2 has been demonstrated to regulate APP levels and processing in the brain, which may modulate AD pathogenesis . When co-expressed with APP, FBXO2 significantly decreases steady-state levels of APP as measured by Western blot .

• Impact on APP processing enzymes: FBXO2 also regulates levels of ADAM10, an enzyme involved in APP processing, with a more pronounced effect on the immature form of ADAM10 that carries high-mannose glycans (the major ligand for FBXO2) .

• Neuronal phenotype in knockout models: Primary cultures of hippocampal neurons from FBXO2-knockout mice show elevated APP levels compared to wild-type neurons . This provides a valuable model system for studying the consequences of FBXO2 deficiency.

To effectively study these mechanisms using FBXO2 antibodies, researchers can:

• Compare FBXO2, APP, and ADAM10 levels in brain tissues from normal versus disease states

• Investigate the interaction between FBXO2 and APP using co-immunoprecipitation with FBXO2 antibodies

• Assess APP processing products (like Aβ) in the presence or absence of FBXO2

• Examine the subcellular localization of FBXO2 in neurons using immunofluorescence

Understanding FBXO2's role in neurodegenerative diseases could potentially lead to novel therapeutic approaches targeting protein quality control mechanisms.

What are the optimal western blot protocols for FBXO2 detection?

Based on the research literature, the following western blot protocol has proven effective for FBXO2 detection:

• Sample preparation:

  • For cell lines like U-87 MG (human glioblastoma-astrocytoma epithelial cells), whole cell lysates at approximately 20 μg protein concentration yield good results .

  • For neuronal samples, primary cultures of hippocampal neurons can be directly lysed and processed for western blot analysis .

• Antibody concentration:

  • Anti-FBXO2 antibody [EPR7328(2)] (ab133717) has been successfully used at a 1/10000 dilution .

  • Secondary antibody selection should match the host species of the primary antibody (typically goat anti-rabbit for rabbit monoclonal antibodies).

• Detection system:

  • Both immature (high-mannose glycan-bearing) and mature (complex glycan-bearing) forms of FBXO2-targeted proteins can be detected on Western blot .

  • When studying FBXO2's effect on proteins like APP and ADAM10, both immature and mature forms should be analyzed, as FBXO2 may differentially affect these forms .

• Controls:

  • Positive control: NeuAc, a known in vitro substrate for FBXO2, can serve as a positive control .

  • Negative control: When studying FBXO2 knockout effects, wild-type cells/tissues should be included for comparison .

• Evaluation:

  • For studying xenophagy, the conversion of LC3-I to LC3-II should be monitored alongside FBXO2 levels to evaluate autophagy induction .

  • For protein degradation studies, steady-state levels of co-expressed substrates can be measured by Western blot to assess FBXO2's effect on protein stability .

When optimizing western blot protocols for FBXO2 detection, researchers should carefully validate antibody specificity and consider the glycosylation status of potential FBXO2 target proteins, as this may influence detection patterns.

How can researchers effectively validate FBXO2 antibody specificity?

Validating FBXO2 antibody specificity is crucial for generating reliable research data. Based on the literature, the following approaches are recommended:

• Genetic validation:

  • Utilize FBXO2-knockout cells/tissues as negative controls .

  • CRISPR/Cas9 genome editing has been successfully used to generate FBXO2-knockout cell lines for antibody validation .

• Functional validation:

  • Test the antibody's ability to detect changes in FBXO2 levels under conditions known to affect FBXO2 expression or activity.

  • Assess co-localization with known FBXO2 interaction partners or substrates .

• Specificity testing:

  • Test for cross-reactivity with other F-box family members, particularly close homologs like FBXO6 and FBXO27 .

  • Evaluate specificity across multiple applications (WB, IF, IHC) as performance can vary by application.

• Reproducibility assessment:

  • Verify antibody performance across different lots and sources.

  • Ensure consistent results across independent experiments and researchers.

• Multi-antibody approach:

  • When possible, use multiple antibodies targeting different epitopes of FBXO2.

  • Compare staining/detection patterns between different antibodies to confirm specificity.

For commercial antibodies like anti-FBXO2 antibody [EPR7328(2)] (ab133717), reviewing the manufacturer's validation data and published citations can provide valuable information on specificity and optimal usage conditions .

What are the key considerations for immunofluorescence studies of FBXO2 in xenophagy research?

Immunofluorescence studies have revealed critical insights into FBXO2's role in xenophagy. The following considerations are important when designing such experiments:

• Temporal dynamics:

  • FBXO2 recruitment to bacteria occurs with specific timing - FBXO2 is recruited to GAS more frequently than LC3 at 2 and 4 hours after infection .

  • Time course experiments should be designed to capture these dynamics.

• Co-localization analysis:

  • Multiple markers should be used to analyze the spatial relationship between FBXO2 and other components of the xenophagy pathway.

  • Signal-intensity plots of FBXO2 and LC3 can reveal that signal peaks of FBXO2 are inside LC3-positive circles, suggesting FBXO2 targets bacteria rather than LC3 vacuoles .

• Bacterial mutant controls:

  • Wild-type bacteria should be compared with mutants lacking specific surface structures.

  • Δslo GAS (lacking streptolysin O) can be used to demonstrate that FBXO2 translocation occurs specifically upon cytosolic escape of bacteria .

  • ΔgacI GAS (lacking GlcNAc residues) helps confirm that GlcNAc is involved in targeting FBXO2 to bacteria .

• Quantification approaches:

  • The percentage of infected cells exhibiting FBXO2-positive bacteria should be quantified (>50% for FBXO2 and >30% for the related protein FBXO6) .

  • Both recruitment frequency and signal intensity should be measured.

• Technical considerations:

  • EmGFP-tagged FBXO2 has been successfully used to visualize FBXO2 recruitment to bacteria .

  • Confocal microscopy is recommended for accurate assessment of co-localization.

By carefully considering these factors, researchers can design robust immunofluorescence experiments to study FBXO2's role in xenophagy against bacterial pathogens.

What are the most common challenges when using FBXO2 antibodies in different applications?

Researchers working with FBXO2 antibodies may encounter several challenges that can impact experimental outcomes:

• Specificity concerns:

  • FBXO2 belongs to a family of related F-box proteins, including FBXO6 and FBXO27, which share structural similarities . This can lead to potential cross-reactivity.

  • Solution: Validate antibody specificity using FBXO2-knockout controls and compare reactivity against related family members.

• Detection of different glycoforms:

  • FBXO2 targets proteins with high-mannose glycans, and the glycosylation status of target proteins can affect binding .

  • Solution: Consider using glycosidase treatments as controls to verify glycan-dependent interactions.

• Expression level variations:

  • FBXO2 expression levels can vary significantly between tissues and cell types, with highest expression typically in brain tissue .

  • Solution: Optimize antibody dilutions for each tissue/cell type and include positive controls from tissues known to express FBXO2.

• Epitope masking in complex formation:

  • FBXO2 functions as part of the SCF complex with SKP1, CUL1, and ROC1 , which could potentially mask antibody epitopes.

  • Solution: Try multiple antibodies targeting different regions of FBXO2 or use epitope retrieval methods.

• Temporal dynamics in functional studies:

  • FBXO2 recruitment to targets (e.g., bacteria in xenophagy) follows specific temporal patterns .

  • Solution: Design time-course experiments with appropriate intervals to capture the dynamic nature of FBXO2 interactions.

By anticipating these challenges and implementing appropriate controls and optimization steps, researchers can enhance the reliability and reproducibility of their FBXO2 antibody-based experiments.

How can researchers optimize immunohistochemistry protocols for FBXO2 detection in tissue samples?

Optimizing immunohistochemistry (IHC) protocols for FBXO2 detection is essential, particularly for clinical and prognostic studies in tissues like colorectal cancer:

• Sample preparation:

  • Formalin-fixed, paraffin-embedded (FFPE) tissue samples have been successfully used for FBXO2 IHC .

  • Optimal section thickness is typically 4-5 μm.

• Antigen retrieval:

  • Heat-induced epitope retrieval methods are generally recommended, as FBXO2 epitopes may be masked during fixation.

  • Specific buffer conditions should be optimized based on the antibody manufacturer's recommendations.

• Blocking and antibody incubation:

  • Adequate blocking of endogenous peroxidase activity and non-specific binding is crucial.

  • Primary antibody dilution should be optimized for each antibody and tissue type, with incubation time typically ranging from 1 hour at room temperature to overnight at 4°C.

• Detection system:

  • Secondary antibody selection should match the host species of the primary antibody.

  • For clinical studies, polymer-based detection systems often provide optimal signal-to-noise ratios.

• Scoring and analysis:

  • Establish a standardized scoring system for FBXO2 expression (e.g., positive vs. negative, or semi-quantitative scales) .

  • Include positive and negative controls in each IHC run to ensure consistency.

• Validation:

  • Consider using a tissue microarray (TMA) approach for initial optimization across multiple samples.

  • When studying FBXO2 in cancer tissues, correlation with other markers like Ki67 and N-cadherin can provide valuable context .

Statistical analysis of IHC results should be performed using appropriate methods such as Chi-squared tests for correlations with clinicopathological factors and Kaplan-Meier survival analysis for prognostic studies .

How should researchers interpret conflicting data on FBXO2 function across different experimental systems?

When encountering conflicting data on FBXO2 function across different experimental systems, researchers should consider the following analytical approaches:

• Biological context specificity:

  • FBXO2 functions in multiple pathways, including ERAD , xenophagy , and regulation of specific substrates like APP .

  • Apparent contradictions may reflect genuine biological differences in FBXO2 function across tissues, cell types, or pathological states.

  • Solution: Clearly define the biological context of each study and avoid overgeneralizing findings.

• Methodological variations:

  • Different antibodies, detection methods, and experimental conditions can yield divergent results.

  • Solution: Standardize protocols when comparing across studies and validate key findings using multiple methodological approaches.

• Substrate specificity considerations:

  • FBXO2 recognizes high-mannose glycans , and its substrate specificity may vary depending on glycosylation patterns.

  • Solution: Characterize glycosylation status of putative FBXO2 substrates in each experimental system.

• Temporal dynamics:

  • FBXO2 functions in dynamic processes such as protein degradation and xenophagy, with distinct temporal patterns .

  • Solution: Implement time-course analyses rather than single time-point measurements.

• Model system limitations:

  • Findings in cell lines may not always translate to primary cells or in vivo systems.

  • Solution: Validate key findings across multiple model systems (cell lines, primary cultures, animal models) when possible.

When publishing or presenting FBXO2-related research, clearly acknowledge these potential sources of variation and discuss how they might reconcile apparently conflicting observations across studies.

What emerging research directions are being explored with FBXO2 antibodies?

Based on current literature, several promising research directions utilizing FBXO2 antibodies are emerging:

• Cancer biology and prognostics:

  • FBXO2 expression has shown prognostic significance in colorectal cancer , suggesting potential applications in other malignancies.

  • Future research may explore FBXO2 as part of multi-marker prognostic panels for various cancer types.

• Neurodegenerative disease mechanisms:

  • FBXO2's role in regulating APP levels and processing suggests potential involvement in Alzheimer's disease pathogenesis .

  • Emerging research may investigate FBXO2 manipulation as a therapeutic strategy for neurodegenerative disorders.

• Host-pathogen interactions:

  • FBXO2's role in xenophagy against group A Streptococcus opens new avenues for studying innate immunity .

  • Future studies may explore FBXO2's role in defending against other bacterial pathogens or even viruses.

• Glycoprotein quality control:

  • FBXO2's function in recognizing and facilitating degradation of proteins with high-mannose glycans suggests broader applications in studying protein quality control mechanisms.

  • Research may expand to investigating FBXO2's role in diseases characterized by protein misfolding or aggregation.

• SCF complex dynamics:

  • FBXO2 functions as part of the SCF complex with SKP1, CUL1, and ROC1 .

  • Future research may explore how FBXO2 is regulated within this complex and how the composition of the complex affects substrate selection.

These emerging directions highlight the versatility of FBXO2 antibodies as tools for investigating diverse biological processes and disease mechanisms.

How do the functions of FBXO2 compare with other F-box family members in experimental systems?

The F-box protein family contains numerous members with diverse functions. Comparative analysis of FBXO2 with other family members yields important insights:

Key comparative insights:

When designing experiments to study F-box proteins, researchers should consider these functional similarities and differences, particularly when interpreting phenotypes in knockout systems or when selecting specific antibodies.

What experimental approaches can distinguish between FBXO2 and closely related family members?

Distinguishing between FBXO2 and closely related family members like FBXO6 and FBXO27 is crucial for accurate functional characterization. The following experimental approaches are recommended:

• Antibody-based discrimination:

  • Use highly specific antibodies validated against knockout controls .

  • Perform western blot analysis to confirm antibody specificity against recombinant FBXO2, FBXO6, and FBXO27.

• Functional assays:

  • Exploit differential recruitment patterns to bacteria - FBXO2 and FBXO6 are recruited to intracellular GAS, while FBXO27 is not .

  • Compare ubiquitination patterns mediated by different F-box proteins using specific substrates.

• Genetic approaches:

  • Use CRISPR/Cas9 to generate specific knockouts of individual F-box proteins .

  • Perform rescue experiments with wild-type versus mutant versions of each F-box protein.

  • Implement siRNA-mediated knockdown with carefully designed sequences that avoid cross-targeting.

• Structural analysis:

  • Focus on the sugar-binding motif, which is critical for FBXO2 function in recognizing GlcNAc residues .

  • The Y289A/W290A mutation in FBXO2 affects its sugar-binding capacity and can help distinguish its function from related proteins .

• Subcellular localization:

  • Track different EmGFP-tagged F-box proteins during bacterial infection or other cellular processes .

  • Analyze co-localization patterns with specific cellular compartments or structures.

By combining these approaches, researchers can effectively distinguish the functions of FBXO2 from those of its closely related family members, leading to more precise understanding of their respective biological roles.