FBX8 Antibody

What is FBX8 and why is it important in cancer research?

FBX8 is a novel component of F-box proteins involved in the ubiquitin-dependent proteolytic pathway. It has E3 ligase activity that mediates the ubiquitination of ARF6 (a GTP-binding protein) . Unlike typical ubiquitination, FBX8-mediated ubiquitination of ARF6 does not induce ARF6 degradation but rather inhibits its function .

FBX8 is significant in cancer research because:

• It is downregulated in multiple cancer types, including hepatocellular carcinoma, glioma, and invasive breast cancer cells

• FBX8 downregulation correlates with tumor progression, invasiveness, and poor prognosis

• It acts as an invasion and metastasis suppressor in several cancer types

• Its expression status has been identified as an independent prognostic factor

What tissue expression patterns does FBX8 exhibit?

FBX8 expression varies significantly between normal and cancerous tissues:

What antibodies are available for FBX8 detection?

Based on the literature, several antibody types have been used for FBX8 detection:

• Rabbit polyclonal antibody against recombinant GST-FBX8 protein:

  • Containing amino acids 1-67 of FBX8 fused to the carboxyl-terminus of glutathione-transferase (GST)

  • Affinity-purified using GST-FBX8 protein

• Commercial antibodies: Various commercial sources provide FBX8 antibodies, though specific validation data should be requested from vendors .

What experimental applications are validated for FBX8 antibodies?

The literature reports FBX8 antibodies being successfully used in:

How should I validate an FBX8 antibody for experimental use?

Proper antibody validation is critical for generating reliable research data. For FBX8 antibodies, implement these validation strategies:

• Specificity testing:

  • Western blot analysis to confirm detection at the expected molecular weight

  • Use of knockout (KO) cell lines as negative controls

  • Peptide competition assays to confirm binding specificity

• Application-specific validation:

  • Validate separately for each experimental method (Western blot, IHC, IF)

  • Use positive and negative controls appropriate for each application

• Cross-validation:

  • Compare results using independent antibodies targeting different FBX8 epitopes

  • Compare antibody-based results with orthogonal methods (e.g., mass spectrometry)

Recent recommendations from YCharOS found that 50-75% of commercially available antibodies perform adequately in specific applications, but validation is essential as ~12 publications per protein target included data from antibodies that failed to recognize the relevant target protein .

What controls are critical when working with FBX8 antibodies?

Knockout cell lines provide superior control compared to other methods, particularly for Western blots and immunofluorescence imaging .

What approaches can resolve discrepancies between FBX8 mRNA and protein expression?

In several cancer cell types, FBX8 mRNA is present while protein expression is absent . To investigate this discrepancy:

• Post-transcriptional regulation analysis:

  • Identify microRNAs potentially targeting FBX8 mRNA

  • Examine RNA-binding proteins that might affect translation efficiency

• Protein stability assessment:

  • Treat cells with proteasome inhibitors to determine if FBX8 is rapidly degraded

  • Perform pulse-chase experiments to measure protein half-life

  • Compare ubiquitination status of FBX8 in normal versus cancer cells

• Translational efficiency:

  • Analyze polysome profiling to determine if FBX8 mRNA is efficiently translated

  • Examine potential translational inhibitors specific to FBX8

How can I design experiments to investigate FBX8's role in cancer progression?

Based on published research, a comprehensive experimental approach includes:

• Expression analysis:

  • Compare FBX8 levels between tumor and adjacent normal tissues

  • Correlate expression with clinical parameters (tumor grade, patient survival)

• Functional studies:

  • Gain-of-function: Overexpress FBX8 in cancer cell lines with low endogenous expression

  • Loss-of-function: Use siRNA, shRNA, or CRISPR to knockdown/knockout FBX8

  • Measure effects on proliferation, migration, invasion in vitro

  • Assess tumor growth and metastatic potential in vivo

• Mechanistic investigations:

  • Study FBX8-ARF6 interaction through co-immunoprecipitation

  • Perform ubiquitination assays to assess E3 ligase activity

  • Identify additional FBX8 substrates through mass spectrometry-based approaches

What are the optimal methods for studying FBX8-mediated ubiquitination of ARF6?

To investigate this non-canonical ubiquitination mechanism:

• In vitro ubiquitination assays:

  • Recombinant proteins: GST-FBX8, ARF6, E1, E2, ubiquitin

  • Detection methods: Western blotting with anti-ubiquitin and anti-ARF6 antibodies

• Cell-based ubiquitination assays:

  • Co-transfection of FBX8, ARF6, and tagged ubiquitin

  • Immunoprecipitation of ARF6 followed by detection of ubiquitin modifications

  • Use ubiquitin mutants (K48R, K63R) to determine linkage types

• Functional consequences:

  • ARF6 activity assays (GTP-binding) in presence/absence of FBX8

  • Localization studies to determine if ubiquitination affects ARF6 cellular distribution

  • Invasion/migration assays to correlate ubiquitination with functional outcomes

How can I ensure reproducibility when working with FBX8 antibodies?

The "antibody characterization crisis" has led to an alarming increase in scientific publications containing misleading or incorrect interpretations due to inadequately characterized antibodies . To enhance reproducibility:

• Detailed documentation:

  • Record complete antibody information (supplier, catalog number, lot number, RRID)

  • Maintain detailed validation protocols and results

  • Share methodological details in publications

• Multiple validation approaches:

  • Use orthogonal methods to confirm findings

  • Employ multiple antibodies targeting different epitopes

  • Include appropriate positive and negative controls in every experiment

• Technical considerations:

  • Standardize experimental protocols

  • Implement blinding procedures when scoring or analyzing results

  • Consider automated staining systems to reduce technical variability

What quantification methods are recommended for FBX8 expression analysis?

For reliable quantification:

When reporting FBX8 expression data, clearly describe scoring methods, thresholds for positivity, and include representative images.

How does FBX8 expression correlate with clinical outcomes in cancer patients?

Published studies reveal significant correlations between FBX8 expression and clinical parameters:

These findings suggest FBX8 may serve as a prognostic biomarker in multiple cancer types.

What considerations are important when analyzing FBX8 in patient-derived samples?

For clinical sample analysis:

• Sample preparation:

  • Fixation methods may affect epitope accessibility

  • Compare fresh-frozen vs. FFPE tissue performance

  • Optimize antigen retrieval methods for IHC

• Heterogeneity considerations:

  • Analyze multiple regions within a tumor sample

  • Use tissue microarrays for high-throughput screening

  • Consider intratumoral heterogeneity in expression patterns

• Comprehensive analysis:

  • Correlate FBX8 expression with other molecular markers

  • Integrate with genomic and transcriptomic data

  • Perform survival analyses stratified by FBX8 expression levels