FBXO9 Antibody

Basic Research Questions

• What is FBXO9 and what fundamental functions should researchers consider when selecting antibodies?

FBXO9 functions as a substrate recognition component of the SKP1-cullin-1-RBX1 (SCF) E3 ubiquitin ligase complex, mediating ubiquitination and proteasomal degradation of target proteins. It plays critical roles in multiple cellular processes including cell cycle regulation, cell proliferation, and maintenance of chromosome stability . When selecting antibodies, researchers should consider that FBXO9 contains two major domains: the F-box domain that binds the SCF complex and the TPR domain involved in protein interactions . Different antibodies may target distinct regions, affecting detection of specific protein interactions.

• What applications are FBXO9 antibodies validated for in research settings?

FBXO9 antibodies have been validated for multiple research applications including:

• Western Blotting (WB): Primary application for detecting FBXO9 expression levels

• Immunohistochemistry (IHC): Both paraffin-embedded (IHC-P) and standard protocols

• Immunofluorescence (IF): For subcellular localization studies

• ELISA: For quantitative measurements

• Flow cytometry (FACS): For cell-based assays

Experimental approaches combining these techniques have been crucial in studies revealing FBXO9's contradictory roles in different cancer types .

• What epitope regions do commonly available FBXO9 antibodies target and how does this affect experimental design?

FBXO9 antibodies target various epitope regions including:

• Middle Region (amino acids vary by manufacturer)

• C-Terminal Region (AA 425-447)

• AA 13-111 (N-terminal region)

• AA 346-373 (C-terminal region)

• AA 179-228 (middle region)

• AA 400 to C-terminus

The epitope choice is critical for experimental design. For studying FBXO9's interactions with the SCF complex, antibodies targeting the F-box domain may interfere with complex formation. For studying substrate interactions, antibodies targeting the TPR domain region could block substrate binding. When investigating FBXO9's role in V-ATPase assembly inhibition, researchers should select antibodies that don't interfere with the protein regions involved in HSPA8 interaction .

• What species reactivity profiles are available for FBXO9 antibodies and how should cross-reactivity be validated?

FBXO9 antibodies demonstrate varying reactivity profiles:

• Human-specific antibodies

• Human/Mouse cross-reactive antibodies

• Broadly cross-reactive antibodies covering multiple species including Human, Mouse, Rat, Cow, Dog, Zebrafish, Horse, Pig, Rabbit, Guinea Pig, and even Saccharomyces cerevisiae

Cross-reactivity validation should include positive controls from each species. For example, when studying FBXO9 in mouse models of leukemia , preliminary validation using both human and mouse cell lysates is essential to confirm specificity. Sequence alignment analysis should be performed when the antibody is predicted to react with multiple species to identify potential variations in epitope regions.

• What validation methods should researchers implement when using FBXO9 antibodies?

Comprehensive validation should include:

• Western blot analysis using cell lysates as positive controls

• Knockout/knockdown validation: Compare signals between wild-type cells and FBXO9 knockout/knockdown cells generated using methods described in studies (shRNA, siRNA, or CRISPR-Cas9)

• Immunoprecipitation followed by mass spectrometry to confirm specificity

• For functional studies, validation with multiple antibodies targeting different epitopes

• Testing for cross-reactivity with other F-box family proteins to ensure specificity

Studies using lentiviral vectors carrying miRNA-based shRNA sequences have effectively validated antibody specificity by confirming signal reduction in knockdown cells .

Advanced Research Questions

• How can researchers optimize FBXO9 antibody protocols for detecting both ubiquitinated and non-ubiquitinated forms?

When studying FBXO9's E3 ligase function, detecting both forms requires methodological optimization:

Protocol Recommendations:

• Preserve ubiquitinated forms by including deubiquitinase inhibitors (N-ethylmaleimide, 10-20 mM) in lysis buffers

• Use proteasome inhibitors (MG132 or bortezomib at 10μM for 4-8 hours) before cell collection

• For immunoprecipitation experiments, employ denaturing conditions (1% SDS lysis buffer with subsequent dilution) to disrupt protein-protein interactions

• Utilize antibodies targeting different FBXO9 regions to ensure detection of all forms

• Consider dual immunoprecipitation approaches: first pull down with anti-ubiquitin antibodies, then probe with FBXO9 antibodies

The ubiquitination status of FBXO9 targets like ATP6V1A can significantly impact functional outcomes as demonstrated in lung cancer metastasis studies . For substrates like TEL2 and TTI1, phosphorylation by CK2 is a prerequisite for FBXO9-mediated ubiquitination, necessitating phosphatase inhibitors in buffers .

• How should researchers interpret contradictory findings regarding FBXO9 expression and function across different cancer types?

FBXO9 exhibits context-dependent roles, functioning as:

• A tumor suppressor in lung cancer by inhibiting V-ATPase assembly and reducing vesicular acidification

• An oncogene in hepatocellular carcinoma (HCC) by promoting tumor growth and metastasis

• A tumor suppressor in acute myeloid leukemia (AML), with reduced expression correlating with poorer survival

• An oncogene in multiple myeloma through mTORC signaling modulation

Methodological approach to resolve contradictions:

• Validate FBXO9 expression using multiple antibodies targeting different epitopes

• Conduct tissue-specific expression analysis comparing tumor vs. normal tissue

• Perform substrate identification in each cancer type using immunoprecipitation followed by mass spectrometry

• Analyze tissue-specific interaction partners that may influence FBXO9 function

• Consider post-translational modifications that may alter FBXO9 activity in different cellular contexts

As demonstrated in AML studies, FBXO9 can function in a dose-dependent manner where even heterozygous loss can promote disease progression .

• What experimental controls are essential when studying FBXO9 in cancer models using antibody-based methods?

Essential controls include:

• Expression controls:

  • FBXO9 overexpression and knockdown/knockout validation samples

  • Normal tissue adjacent to tumor samples

  • Panel of cell lines with known FBXO9 expression levels

• Specificity controls:

  • Competitive blocking with immunizing peptide

  • Secondary antibody-only controls

  • Isotype controls for immunohistochemistry

• Functional controls:

  • Proteasome inhibitor-treated samples (e.g., bortezomib at 10-12 nM)

  • Samples with mutated F-box domain to disrupt SCF complex formation

  • Samples with mutated substrate-binding domain

• Animal model controls:

  • Conditional knockout models using systems like Mx1-cre with poly(I:C) induction, parallel to models described in leukemia studies

  • Age-matched and sex-matched wild-type controls

  • Heterozygous FBXO9 knockout models to assess dose-dependent effects

For metastasis studies, controls should include both primary tumors and metastatic nodules, as FBXO9's role may differ between these contexts .

• How can researchers design experiments to investigate FBXO9's interaction with the V-ATPase assembly?

Based on FBXO9's role in inhibiting V-ATPase assembly , the following experimental design is recommended:

Experimental Strategy:

• Protein Interaction Analysis:

  • Co-immunoprecipitation using antibodies targeting different FBXO9 domains and V-ATPase components

  • Proximity ligation assays to visualize FBXO9-ATP6V1A interactions in situ

  • FRET/BRET assays to monitor real-time interactions

• Ubiquitination Analysis:

  • In vitro ubiquitination assays with recombinant FBXO9 and ATP6V1A

  • Ubiquitin pulldown followed by ATP6V1A detection

  • Cycloheximide chase assays with and without proteasome inhibitors

• Functional Assessment:

  • Vesicular acidification assays using acridine orange or LysoTracker in cells with FBXO9 manipulation

  • V-ATPase assembly analysis through glycerol gradient fractionation

  • Subcellular localization studies of ATP6V1A in FBXO9-depleted vs. overexpressing cells

• Validation in Cancer Models:

  • Tissue microarray analysis correlating FBXO9 and ATP6V1A levels

  • Mouse models with peptide inhibitors of ATP6V1A ubiquitination (V1A-23 aa peptide)

  • Assessment of metastatic potential using tail vein injection models

This approach has successfully demonstrated FBXO9's role in regulating V-ATPase assembly through ATP6V1A ubiquitination .

• What methodological considerations are important when using FBXO9 antibodies in studies of proteasome activity?

Given FBXO9's relationship with proteasome activity , researchers should consider:

Methodological Considerations:

• Antibody Selection:

  • Use antibodies that can detect both free and SCF complex-bound FBXO9

  • Consider epitope accessibility in different cellular compartments

• Proteasome Activity Measurement:

  • Implement fluorogenic substrate assays in parallel with FBXO9 detection

  • Use bortezomib dose-response experiments (8-12 nM range) as calibration controls

  • Include time-course analyses to capture dynamic changes

• Sample Processing:

  • Avoid freeze-thaw cycles that may affect proteasome integrity

  • Collect samples at consistent times to control for circadian variations

  • Include ATP in buffers to maintain proteasome stability during preparation

• Data Analysis:

  • Normalize proteasome activity to total protein content

  • Consider cell cycle phase since proteasome activity varies throughout the cycle

  • Analyze correlation between FBXO9 levels and proteasome activity across samples

In AML studies, loss of FBXO9 led to increased proteasome component expression and activity, with IC50 calculations for bortezomib of 10.03 nM in FBXO9-deficient cells compared to 11.76 nM in controls .

• How should researchers select FBXO9 antibodies for studying different functional domains and their interactions?

Domain-specific antibody selection requires careful consideration:

Domain-Specific Selection Strategy:

• F-box Domain Studies (substrate recruitment to SCF):

  • Select antibodies targeting regions outside the F-box domain

  • Avoid antibodies that might interfere with SCF complex formation

  • Include co-immunoprecipitation controls to verify complex assembly

• TPR Domain Studies (protein-protein interactions):

  • Use antibodies targeting the N-terminal or C-terminal regions

  • Consider conformation-specific antibodies if available

  • Validate that the antibody doesn't block interaction sites

• Post-translational Modification Studies:

  • Select antibodies that don't target regions containing potential modification sites

  • Use phospho-specific or ubiquitin-specific antibodies in conjunction with total FBXO9 antibodies

  • Include phosphatase or deubiquitinase treatments as controls

• Structural Studies:

  • For immunoprecipitation followed by structural analysis, select antibodies with high affinity but minimal structural impact

  • Consider using tagged FBXO9 constructs for pull-down when structural integrity is critical

Research on FBXO9's TPR domain has been crucial for understanding its role in substrate recognition, as demonstrated in the conditional knockout mouse model targeting exon 4 which contains most of the TPR domain .

• What protocols are recommended for using FBXO9 antibodies in mouse models of leukemia and other cancers?

Based on leukemia studies using FBXO9 conditional knockout models :

Recommended Protocol:

• Mouse Model Generation:

  • Use conditional knockout approaches (Mx1-cre or tissue-specific Cre)

  • Validate knockout efficiency by immunoblotting with antibodies targeting multiple FBXO9 epitopes

  • Consider heterozygous models to study dose-dependent effects

• Tissue Processing:

  • For bone marrow: Collect samples in PBS with 2% FBS, red blood cell lysis, fixation in 4% PFA

  • For liver/lung metastasis: Perfuse with PBS before collection, fix in 10% formalin

  • For immunohistochemistry: Use antigen retrieval with citrate buffer pH 6.0

• Flow Cytometry:

  • Single-cell suspensions from bone marrow, spleen, or tumor

  • Fix with 2% paraformaldehyde for 10 minutes

  • Permeabilize with 0.1% Triton X-100 for intracellular staining

  • Use 3% goat serum for blocking

• Analysis Recommendations:

  • Include both primary tumors and metastatic sites

  • Perform parallel analyses of FBXO9 and its substrates

  • Correlate FBXO9 levels with proteasome activity and clinical outcomes

For leukemia models, samples from 6 mice per group (as in published studies) provide sufficient statistical power for detecting significant differences in FBXO9 expression and function .

• How should FBXO9 antibody selection and experimental design differ when studying pluripotency versus cancer progression?

Different biological contexts require tailored approaches:

Context-Specific Recommendations:

• Pluripotency Studies:

  • Select antibodies validated in stem cell contexts

  • Include co-staining with pluripotency markers (Oct4, Nanog)

  • Use stem cell-specific lysis buffers that maintain protein-protein interactions

  • Consider chromatin immunoprecipitation protocols to study FBXO9's role in regulating pluripotency genes

  • Include differentiation time-course analyses to track FBXO9 dynamics

• Cancer Progression Studies:

  • Select antibodies validated in relevant cancer tissues

  • Include stage-specific tumor samples to track expression changes

  • Use metastasis models (tail vein injection, orthotopic implantation)

  • Incorporate survival analyses correlating FBXO9 levels with outcome

  • Consider cancer type-specific substrate analyses

• Comparative Analysis Protocol:

  • Standardize detection methods across contexts

  • Use recombinant FBXO9 as control for antibody performance

  • Implement multiplexed approaches to detect FBXO9 along with context-specific markers

  • Include tissue microarrays spanning developmental and malignant tissues