FBXL13 Antibody

What is FBXL13 and what is its main cellular function?

FBXL13 is a binding determinant of SCF (SKP1-CUL1-F-box)-family E3 ubiquitin ligases that is enriched at centrosomes. It functions primarily to regulate centrosome organization and microtubule nucleation capacity through targeted protein degradation. FBXL13 interacts with several centrosomal proteins, including Centrin-2, Centrin-3, CEP152, and CEP192, with CEP192 being specifically targeted for proteasomal degradation . This regulation impacts centrosome microtubule nucleation activity and affects cell motility, suggesting potential implications in tumor development and progression. FBXL13 acts as a fine-tuner of CEP192 levels to maintain steady-state centrosome microtubule nucleation activity .

How does FBXL13 regulate centrosome function and cell motility?

FBXL13 regulates centrosome function through the specific ubiquitin-mediated proteolysis of CEP192 isoform 3. When FBXL13 is overexpressed, it leads to reduced levels of centrosomal CEP192 and γ-tubulin, which disrupts centrosomal microtubule arrays . This disruption results in altered cell morphology consistent with a compromised microtubule network. Conversely, depletion of FBXL13 causes increased levels of CEP192 and γ-tubulin at centrosomes, leading to defects in cell motility .

The relationship between FBXL13 and cell motility appears to be biphasic: increased levels of FBXL13 enhance cell motility by reducing centrosomal microtubule nucleation capacity and causing hyperpolarization, while reduced FBXL13 levels promote centrosomal microtubule nucleation at the expense of extracentrosomal microtubules, resulting in decreased migration .

What epitopes should researchers target when selecting FBXL13 antibodies?

When selecting antibodies against FBXL13, researchers should consider targeting:

• The F-box domain, which is critical for interaction with SKP1 and formation of functional SCF complexes

• The leucine-rich repeat regions, which are involved in substrate recognition

• Isoform-specific regions, particularly for distinguishing between FBXL13 isoform 1 and isoform 3

The choice of epitope should be guided by the specific research question. For studying general FBXL13 function, antibodies targeting conserved regions are preferable. For isoform-specific studies, targeting the variable carboxyl-terminal regions is recommended, as these regions account for the ~30% difference in interacting proteins between isoforms .

How should researchers validate FBXL13 antibody specificity for immunofluorescence studies?

Validating FBXL13 antibody specificity for immunofluorescence requires a multi-step approach:

• RNAi validation: Perform siRNA or shRNA-mediated depletion of FBXL13 and confirm reduced immunofluorescence signal. The search results demonstrate effective FBXL13 depletion using both siRNA and shRNA approaches that can be adapted for validation .

• Overexpression controls: Compare staining patterns in cells expressing exogenous FBXL13 (wild-type or tagged versions) with endogenous staining patterns. The search results show that both endogenous and exogenous FBXL13 localize to centrosomes, providing a clear positive control .

• Peptide competition assay: Pre-incubate the antibody with the immunizing peptide to confirm signal abolishment.

• Comparison with multiple antibodies: Use antibodies targeting different epitopes of FBXL13 to confirm consistent staining patterns.

• Co-localization with known interactors: Confirm that FBXL13 co-localizes with known centrosomal markers like Centrin-2, Centrin-3, CEP152, and CEP192 .

What is the optimal protocol for immunoprecipitating FBXL13 and its binding partners?

Based on the methodologies described in the search results, the following protocol is recommended for immunoprecipitating FBXL13 and its binding partners:

Lysis buffer composition:

• 50 mM HEPES pH 7.2

• 150 mM NaCl

• 0.1% NP-40

• 1% glycerol

• Supplemented with: 1 mM DTT, 20 mM β-glycerophosphate, 0.1 mM PMSF, 20 nM okadaic acid, and complete protease inhibitor cocktail (1:500 dilution)

Protocol for endogenous FBXL13 immunoprecipitation:

• Resuspend cell pellets in three volumes of fresh lysis buffer and incubate on ice for 10 minutes

• Remove debris by centrifugation at 20,000 rcf for 10 minutes

• Incubate lysates with 4 μg of FBXL13 antibody or control IgG overnight at 4°C on a rotator

• Capture antibodies with rProtein A Sepharose Fast Flow beads for 1 hour at 4°C

• Wash samples three times in lysis buffer

• Resuspend in Laemmli buffer and boil at 90°C for 5 minutes before SDS-PAGE analysis

For detection of specific interactions, co-immunoprecipitation can be performed with antibodies against known binding partners such as CEP192, CEP152, Centrin-2, and Centrin-3 .

How can researchers effectively study FBXL13-mediated ubiquitination of CEP192?

To study FBXL13-mediated ubiquitination of CEP192, researchers should employ the following approaches:

• In vivo ubiquitination assays:

  • Express HA-tagged ubiquitin in cells with or without FBXL13 depletion

  • Immunoprecipitate CEP192 (particularly isoform 3) under denaturing conditions to avoid co-immunoprecipitated proteins

  • Detect ubiquitinated species by western blotting with anti-HA antibodies

• Functional mutant analysis:

  • Compare ubiquitination using wild-type FBXL13 versus an F-box deletion mutant (FBXL13 ΔF-box)

  • The F-box deletion mutant retains CEP192 binding capability but cannot recruit the SCF complex, providing an excellent negative control

• Proteasome inhibition:

  • Include conditions with proteasome inhibitors (e.g., MG132) to prevent degradation of ubiquitinated CEP192

  • This approach has been shown to rescue the CEP192 reduction observed upon FBXL13 expression

• Specific substrate validation:

  • Focus on CEP192 isoform 3, as the search results indicate this is the specific target of FBXL13-mediated degradation

  • Use isoform-specific regions or antibodies when possible to distinguish between CEP192 isoforms

What techniques are most effective for studying FBXL13's role in centrosomal microtubule nucleation?

To effectively study FBXL13's role in centrosomal microtubule nucleation, researchers should employ these advanced techniques:

• Quantitative immunofluorescence microscopy:

  • Measure centrosomal CEP192 and γ-tubulin levels following FBXL13 manipulation

  • Use confocal microscopy with appropriate centrosomal markers for co-localization

• Microtubule regrowth assays:

  • Completely depolymerize microtubules using cold treatment or nocodazole

  • Allow microtubule regrowth and quantify centrosomal microtubule arrays

  • Compare cells with normal, elevated, or depleted FBXL13 levels

• Live-cell imaging of microtubule dynamics:

  • Use fluorescently tagged tubulin to visualize microtubule growth in real-time

  • Track microtubule nucleation rate, growth velocity, and catastrophe frequency

• Centrosome isolation and functional assays:

  • Isolate centrosomes from cells with different FBXL13 expression levels

  • Perform in vitro microtubule nucleation assays with purified centrosomes

• Super-resolution microscopy:

  • Apply techniques like STORM or STED to visualize the precise localization of FBXL13 relative to centrosomal components

The search results demonstrate that cells overexpressing FBXL13 have significantly reduced centrosomal γ-tubulin and corresponding reduction in centrosomal microtubule arrays, while FBXL13 depletion results in increased γ-tubulin intensity .

How can researchers investigate the relationship between FBXL13 expression and cancer cell phenotypes?

To investigate the relationship between FBXL13 expression and cancer cell phenotypes, researchers should consider these approaches:

The search results suggest that FBXL13 gain-of-function may be oncogenic, with amplification observed in various cancer types and evidence that FBXL13 knockout downregulates proliferation in patient-derived glioblastoma cells .

Why might FBXL13 antibodies show inconsistent centrosomal staining patterns?

Inconsistent centrosomal staining patterns with FBXL13 antibodies may occur for several reasons:

• Isoform specificity: FBXL13 has multiple isoforms with variable carboxyl-terminal regions. The search results indicate FBXL13-1 and FBXL13-3 share only ~30% overlap in their interacting proteins, suggesting structural differences that could affect antibody recognition .

• Cell cycle-dependent localization: FBXL13's centrosomal localization or interaction with centrosomal proteins might vary throughout the cell cycle, similar to other centrosomal regulators.

• Fixation sensitivity: Different fixation methods can affect epitope accessibility, particularly for centrosomal proteins. Paraformaldehyde fixation may yield different results compared to methanol fixation.

• Antibody penetration issues: The centrosome is a dense structure, and antibody accessibility may be limited without proper permeabilization.

• Post-translational modifications: FBXL13 could undergo modifications that mask epitopes or alter localization.

To address these issues, researchers should:

• Test multiple fixation and permeabilization protocols

• Validate antibody specificity using knockdown controls

• Consider cell cycle synchronization when analyzing centrosomal proteins

• Use multiple antibodies targeting different regions of FBXL13

What controls are essential when studying FBXL13-mediated protein degradation?

When studying FBXL13-mediated protein degradation, the following controls are essential:

• F-box deletion mutant control:

  • Include the FBXL13 ΔF-box mutant, which can bind CEP192 but cannot recruit the SCF complex

  • This control helps distinguish between binding and functional ubiquitination/degradation effects

• Proteasome inhibition:

  • Include conditions with proteasome inhibitors (e.g., MG132)

  • This confirms that the observed protein reduction is due to proteasomal degradation rather than other mechanisms

• Isoform-specific controls:

  • When studying CEP192 degradation, distinguish between isoforms

  • The search results show that FBXL13 specifically targets CEP192 isoform 3 but not isoforms 1 or 2

• RNAi validation:

  • Use multiple siRNA or shRNA sequences targeting FBXL13

  • The search results demonstrate effective validation using two distinct siRNA oligos and shRNA approaches

• Cell cycle controls:

  • Monitor cell cycle profiles to ensure effects are not due to indirect cell cycle alterations

  • The search results confirm that FBXL13 overexpression did not affect cell cycle profiles in HEK293T cells

• Protein half-life measurements:

  • Perform cycloheximide chase experiments to determine the effect on protein stability

  • The search results show this technique effectively demonstrated that FBXL13 reduced CEP192 half-life but not that of Centrin-2 and Centrin-3

How might FBXL13 antibodies be utilized to study centrosome abnormalities in cancer?

FBXL13 antibodies can be leveraged to study centrosome abnormalities in cancer through several innovative approaches:

• Multiplex immunohistochemistry/immunofluorescence:

  • Use FBXL13 antibodies alongside markers for centrosome amplification (e.g., γ-tubulin, Pericentrin)

  • Analyze patient tumor samples to correlate FBXL13 expression with centrosome abnormalities

  • The search results indicate FBXL13 is frequently amplified in multiple cancer types, making this a clinically relevant approach

• Single-cell analysis of heterogeneity:

  • Apply FBXL13 antibodies in single-cell protein analysis methods

  • Identify subpopulations within tumors with altered FBXL13 expression

• Proximity ligation assays:

  • Combine FBXL13 antibodies with antibodies against known interactors

  • Visualize and quantify protein interactions in situ in different cancer cell types

  • This approach would build on the interaction studies described in the search results

• Correlation with invasive phenotypes:

  • Develop tissue microarrays with invasion front samples

  • Analyze FBXL13 expression in relation to local invasion and metastatic potential

  • The search results suggest FBXL13 promotes cell motility, potentially contributing to invasion

What methods can be used to investigate potential therapeutic targeting of the FBXL13-CEP192 pathway?

To investigate potential therapeutic targeting of the FBXL13-CEP192 pathway, researchers can employ these methods:

• Small molecule screening:

  • Develop high-throughput screens for compounds that modulate FBXL13-CEP192 interaction

  • Utilize the direct binding region identified in the search results (CEP192 amino acids 1-630)

• Protein-protein interaction inhibitors:

  • Design peptides or small molecules targeting the FBXL13-CEP192 interface

  • Validate with the in vitro binding assays described in the search results

• Structure-based drug design:

  • Perform crystallographic or cryo-EM studies of the FBXL13-CEP192 complex

  • Use structural information to design targeted inhibitors

• Degrader technology:

  • Develop PROTACs (Proteolysis Targeting Chimeras) or molecular glues

  • Target FBXL13 for degradation in cancers where it is overexpressed

• Combination therapy assessment:

  • Test FBXL13-CEP192 pathway modulation in combination with:

    • Microtubule-targeting chemotherapeutics

    • Mitotic inhibitors

    • Centrosome-targeting drugs

• Synthetic lethality approaches:

  • Identify genes that, when inhibited alongside FBXL13 modulation, lead to cancer cell death

  • Focus on centrosome pathway components and cell migration regulators

The search results suggest that FBXL13's role in promoting cell motility and its frequent amplification in various cancers make it a potential therapeutic target, particularly for inhibiting metastasis .