FBXW2 Antibody

What is FBXW2 and what is its function in normal cellular processes?

FBXW2 (F-box and WD repeat domain containing 2) functions as a substrate recognition component of the SCF (Skp1-Cullin-F-box) E3 ubiquitin ligase complex. This protein contains an N-terminal F-box motif and a C-terminal domain consisting of five WD repeats that are critical for substrate recognition .

The primary function of FBXW2 is to bind specific target proteins and facilitate their ubiquitylation, which marks them for proteasomal degradation. This process is essential for maintaining protein homeostasis and regulating various cellular pathways. FBXW2 recognizes its substrates primarily through a consensus degron motif (TSXXXS), which has been defined in recent research .

Multiple studies have demonstrated that FBXW2 expression is frequently downregulated in various cancer types compared to adjacent normal tissues:

These findings collectively suggest that FBXW2 functions as a tumor suppressor, and its loss may contribute to cancer progression and metastasis.

What are the optimal conditions for Western blot detection of FBXW2 protein?

For successful Western blot detection of FBXW2, researchers should consider the following parameters:

• Antibody Selection and Dilution: Commercial FBXW2 antibodies typically work at dilutions of 1:500-1:2000 for Western blotting . Specifically:

  • Proteintech antibody (11499-1-AP) is recommended at 1:500-1:1000 dilution

  • Boster Bio antibody (A11297-2) works at 1:500-2000 dilution

• Expected Molecular Weight: FBXW2 is typically detected at 43-52 kDa. The calculated molecular weight is 44-52 kDa, while the observed molecular weight is often reported as 52 kDa or 43 kDa . This information is crucial for proper identification of FBXW2 bands.

• Sample Preparation: Based on the research protocols used in the literature, cell lysates should be prepared in denaturing conditions. Several studies successfully detected FBXW2 in various cell lines including HEK-293, PC3, AGS, and H1299 cells .

• Incubation Conditions: Primary antibody incubation is typically performed overnight at 4°C .

• Positive Controls: HEK-293 cells have been validated as a positive control for FBXW2 Western blotting .

How can I optimize immunohistochemistry protocols for detecting FBXW2 in tissue samples?

For effective immunohistochemical detection of FBXW2 in tissue samples, consider these recommendations:

• Antibody Dilution: For IHC applications, a dilution range of 1:50-1:500 is typically recommended . Specifically:

  • Proteintech antibody (11499-1-AP) works at 1:50-1:500 dilution for IHC

  • Boster Bio antibody (A11297-2) is recommended at 1:50-400 for IHC

• Antigen Retrieval: For optimal results, antigen retrieval with TE buffer pH 9.0 is suggested. Alternatively, citrate buffer pH 6.0 may be used .

• Positive Control Tissues: Human colon cancer tissue has been validated as a positive control for FBXW2 IHC .

• Detection System: Standard avidin-biotin complex (ABC) or polymer-based detection systems can be used according to laboratory protocols.

• Quantification: For correlation studies, H-score or IHC scoring systems that account for both staining intensity and percentage of positive cells should be employed, as these approaches were used in studies correlating FBXW2 expression with clinical outcomes .

What controls are necessary when studying FBXW2-mediated protein degradation?

When investigating FBXW2-mediated protein degradation, several controls are essential:

• Wild-type vs. Mutant FBXW2: Include both wild-type FBXW2 and F-box deletion mutant (FBXW2-ΔF) in experiments. The FBXW2-ΔF mutant lacks F-box domain functionality and serves as a negative control for ubiquitylation activity .

• Substrate Phosphorylation Mutants: For studying the role of substrate phosphorylation, include both phosphorylation-mimicking mutants (e.g., β-catenin-3D) and phosphorylation-dead mutants (e.g., β-catenin-3A) of the target protein .

• Proteasome Inhibitors: Include treatment with proteasome inhibitors (e.g., MG132) to confirm that observed protein degradation is proteasome-dependent.

• Half-life Measurements: Perform cycloheximide chase experiments to measure the half-life of target proteins in the presence and absence of FBXW2 to confirm effects on protein stability .

• Ubiquitylation Assays: For in vivo and in vitro ubiquitylation assays, include proper controls such as ubiquitin mutants and His-tagged ubiquitin to confirm specific polyubiquitylation .

How can I design experiments to investigate novel FBXW2-substrate interactions?

To identify and characterize new FBXW2 substrates, consider this systematic approach:

• Substrate Candidate Identification:

  • Perform affinity purification-mass spectrometry with tagged wild-type FBXW2 to identify potential binding proteins .

  • Search for the consensus FBXW2 degron sequence (TSXXXS) in potential candidates using bioinformatics tools .

• Validation of Physical Interaction:

  • Conduct co-immunoprecipitation experiments with both exogenous (overexpressed) and endogenous proteins .

  • Include both wild-type FBXW2 and F-box deletion mutants as controls.

  • Use known FBXW2 substrates (e.g., β-catenin, EGFR) as positive controls .

• Functional Validation:

  • Perform expression correlation analysis: FBXW2 overexpression should reduce substrate levels, while FBXW2 knockdown should increase them .

  • Measure protein half-life using cycloheximide chase assays in FBXW2-manipulated cells.

  • Examine mRNA levels to confirm post-transcriptional regulation .

• Ubiquitylation Assays:

  • Conduct in vivo ubiquitylation assays using His-tagged ubiquitin and appropriate controls.

  • Perform in vitro ubiquitylation assays with purified components to confirm direct effects .

• Degron Motif Identification:

  • Create site-directed mutants of potential phosphorylation sites within the candidate substrate.

  • Test binding and degradation efficiency with these mutants to identify the critical degron motif .

How does phosphorylation affect FBXW2-substrate recognition and what kinases are involved?

Phosphorylation plays a crucial role in FBXW2-substrate recognition through several mechanisms:

• Substrate Phosphorylation:

  • For β-catenin, EGF-AKT1-mediated phosphorylation on Ser552 facilitates FBXW2 binding and subsequent ubiquitylation and degradation .

  • FBXW2 promotes polyubiquitylation of wild-type β-catenin and phosphorylation-mimicking mutant β-catenin-3D, but not the phosphorylation-dead mutant β-catenin-3A .

• Kinases Involved:

  • AKT1 (RAC-alpha serine/threonine-protein kinase 1) is a key kinase that phosphorylates β-catenin following EGF stimulation .

  • In breast cancer, AKT kinase phosphorylates Moesin at Thr-558, which prevents its degradation by FBXW2 by weakening the association between FBXW2 and Moesin .

  • GSK3β has been reported to facilitate FBW2 recognition of other substrates like GCM1 .

• Experimental Approach:

  • To study phosphorylation-dependent interactions, use phosphorylation site mutants (alanine substitutions to prevent phosphorylation or acidic residue substitutions to mimic phosphorylation).

  • Employ kinase inhibitors specific to suspected kinases (e.g., AKT inhibitors) to confirm their roles.

  • Use phospho-specific antibodies to detect phosphorylated forms of the substrate.

• Paradoxical Effects:

  • Interestingly, phosphorylation can have opposite effects on FBXW2-substrate interactions depending on the substrate. For β-catenin, phosphorylation promotes FBXW2 binding and degradation , while for Moesin, AKT-mediated phosphorylation prevents FBXW2-mediated degradation .

What approaches can be used to study the role of FBXW2 in cancer metastasis?

To investigate the role of FBXW2 in cancer metastasis, researchers should employ a multi-faceted approach:

• In Vitro Migration and Invasion Assays:

  • Transwell migration and invasion assays have been successfully used to demonstrate that FBXW2 knockdown promotes invasion of cancer cells, while FBXW2 overexpression inhibits it .

  • Colony formation assays can assess the long-term effects of FBXW2 manipulation on cancer cell growth .

• Molecular Mechanism Studies:

  • Examine the effect of FBXW2 on the expression of metastasis-related genes, particularly matrix metalloproteinases (MMPs) .

  • Assess the nuclear translocation of β-catenin in FBXW2-manipulated cells using nuclear and cytoplasmic fractionation followed by Western blotting .

  • Investigate FBXW2's impact on epithelial-mesenchymal transition (EMT) markers.

• In Vivo Metastasis Models:

  • Tail vein injection models can be used to assess lung metastasis formation in mice with FBXW2-manipulated cancer cells .

  • Orthotopic implantation models provide insights into spontaneous metastasis.

  • Monitor metastasis using bioluminescence imaging of luciferase-expressing cells.

• Clinical Correlation Studies:

  • Analyze FBXW2 expression in primary tumors versus metastatic lesions using tissue microarrays.

  • Correlate FBXW2 expression with clinical parameters including TNM stage, lymph node metastasis, and patient survival .

  • Perform multivariate analysis to determine if FBXW2 is an independent prognostic factor.

• Therapeutic Implications:

  • Investigate whether restoration of FBXW2 expression or function can inhibit metastasis in preclinical models.

  • Explore combinatorial approaches targeting FBXW2 substrates, particularly in contexts where FBXW2 is downregulated.

How can I validate the specificity of FBXW2 antibodies for my experimental applications?

Ensuring antibody specificity is crucial for reliable results. Here are comprehensive validation strategies:

• Genetic Controls:

  • Use FBXW2 knockdown (shRNA or siRNA) and overexpression systems to confirm that the antibody signal changes accordingly in Western blot, IHC, or immunofluorescence applications .

  • CRISPR/Cas9-mediated FBXW2 knockout cells provide the most stringent negative control.

• Antibody Validation Techniques:

  • Perform peptide competition assays where the antibody is pre-incubated with the immunizing peptide before application to samples.

  • Test multiple antibodies targeting different epitopes of FBXW2 to confirm consistent results.

  • Compare results from monoclonal and polyclonal antibodies when available.

• Signal Verification:

  • Confirm that the antibody detects a protein of the expected molecular weight (43-52 kDa for FBXW2) .

  • For tagged constructs, perform parallel detection with both FBXW2 antibody and tag-specific antibody (e.g., FLAG, HA) .

• Cross-Reactivity Assessment:

  • Test the antibody on samples from multiple species if cross-reactivity is claimed (human, mouse, rat) .

  • Include related F-box proteins (e.g., FBXW7, FBXW11) as specificity controls.

• Application-Specific Controls:

  • For IHC, include known positive control tissues (e.g., human colon cancer tissue) and negative controls (primary antibody omission).

  • For IP experiments, include IgG control and input samples.

What factors might affect FBXW2 expression or function in experimental systems?

Several factors can influence FBXW2 expression and function, which researchers should consider when designing experiments:

• Cell Type and Tissue Context:

  • FBXW2 expression varies across different cancer types, with some showing upregulation and others downregulation .

  • Consider the endogenous levels of FBXW2 substrates and regulatory proteins in your experimental system.

• Growth Factors and Signaling Pathways:

  • EGF stimulation activates AKT1, which phosphorylates β-catenin, affecting its interaction with FBXW2 .

  • The AKT-Moesin-SKP2 oncogenic axis interacts with FBXW2 function in breast cancer .

  • Monitor the activation status of these pathways in your experimental system.

• Experimental Manipulations:

  • Stress conditions, serum starvation, or confluency can affect protein degradation pathways.

  • Cell synchronization may impact ubiquitin-proteasome system activity.

  • Proteasome inhibitors (e.g., MG132) should be used carefully as they can have global effects on protein homeostasis.

• Technical Considerations:

  • FBXW2 antibody storage and handling can affect detection sensitivity. Store according to manufacturer recommendations (typically at -20°C with 50% glycerol) .

  • For recombinant expression, consider using inducible systems to minimize selection for cells that tolerate high FBXW2 levels.

  • When studying FBXW2-substrate interactions, co-expression levels should be carefully titrated.

• Genetic Background:

  • Mutations in FBXW2 or its substrates might affect interactions and subsequent degradation.

  • Check cell line databases for known mutations in your experimental system.

How can I interpret contradictory results regarding FBXW2 function in different cancer contexts?

When faced with apparently contradictory results regarding FBXW2 function, consider these factors for interpretation:

• Substrate Specificity:

  • FBXW2 targets different substrates in different cancer types (EGFR in prostate cancer , β-catenin in lung and gastric cancer , Moesin in breast cancer ).

  • The relative importance of each substrate may vary depending on cellular context.

• Regulatory Mechanisms:

  • Phosphorylation can have opposite effects on FBXW2-substrate interactions depending on the substrate and context. For β-catenin, phosphorylation promotes FBXW2 binding , while for Moesin, phosphorylation prevents FBXW2-mediated degradation .

  • The balance of kinases (like AKT) and phosphatases in different cell types may determine net outcomes.

• Experimental Approach:

  • Different knockdown or overexpression strategies may achieve different levels of FBXW2 modulation.

  • Transient versus stable expression systems may yield different results due to compensation mechanisms.

  • In vivo versus in vitro systems may not always agree due to the complexity of the tumor microenvironment.

• Data Analysis:

  • Carefully evaluate the statistical methods used in different studies.

  • Consider the sample sizes and whether appropriate controls were included.

  • Assess whether patient cohorts are comparable across studies (cancer stage, treatment history, etc.).

• Integration Approach:

  • Develop a unifying model that accounts for different observations by considering the specific pathways and contexts studied.

  • Consider that FBXW2 may play different roles at different stages of cancer progression.

  • Collaborate with researchers using different models to directly compare results under standardized conditions.

How might FBXW2 be therapeutically targeted in cancer treatment strategies?

Based on FBXW2's tumor suppressive functions, several therapeutic strategies could be explored:

• Restoring FBXW2 Expression:

  • Investigate epigenetic mechanisms that might silence FBXW2 in cancers, potentially using HDAC inhibitors or DNA methyltransferase inhibitors to restore expression.

  • Explore gene therapy approaches to reintroduce FBXW2 in tumors with low expression.

• Targeting FBXW2 Substrates:

  • In cancers with low FBXW2 expression, directly target the accumulated oncogenic substrates:

    • EGFR inhibitors in prostate cancer contexts with low FBXW2

    • Wnt/β-catenin pathway inhibitors in gastric and lung cancers with FBXW2 deficiency

    • AKT inhibitors to disrupt the AKT-Moesin-SKP2 axis in breast cancer

• Enhancing FBXW2 Activity:

  • Develop small molecules that enhance FBXW2 binding to substrates or increase its catalytic efficiency.

  • Identify compounds that stabilize FBXW2 protein or increase its expression.

• Combination Strategies:

  • Combine FBXW2-targeting approaches with conventional chemotherapy or radiotherapy.

  • Explore synergistic effects with other targeted therapies based on the specific cancer context.

• Biomarker Development:

  • Use FBXW2 expression levels as biomarkers for patient stratification in clinical trials.

  • Explore the ratio of FBXW2 to its substrates (e.g., FBXW2/β-catenin ratio) as a more precise predictive marker .

What are the latest techniques for studying the dynamics of FBXW2-mediated protein degradation?

Recent technological advances have expanded the toolkit for studying ubiquitin-mediated protein degradation:

• Live-Cell Imaging Approaches:

  • Fluorescent protein fusion reporters for real-time monitoring of substrate levels and localization.

  • FRET/BRET-based sensors to detect FBXW2-substrate interactions and ubiquitylation events in living cells.

  • Photoactivatable or photoconvertible fusion proteins to track protein degradation kinetics.

• Proximity-Based Labeling:

  • BioID or TurboID fusion proteins to identify proteins in proximity to FBXW2 in living cells.

  • APEX-based approaches for temporal control of labeling the FBXW2 interactome.

• Advanced Proteomics:

  • Tandem mass tag (TMT) or SILAC approaches to quantitatively profile changes in the proteome upon FBXW2 manipulation.

  • Ubiquitin remnant profiling to identify ubiquitylation sites on FBXW2 substrates.

  • Thermal proteome profiling to identify proteins stabilized or destabilized by FBXW2.

• CRISPR-Based Screens:

  • Genome-wide CRISPR screens to identify genes that modify FBXW2-dependent phenotypes.

  • CRISPR activation/inhibition screens to identify regulators of FBXW2 expression.

• Structural Biology Approaches:

  • Cryo-EM analysis of the SCF-FBXW2 complex with substrates to understand structural determinants of specificity.

  • Hydrogen-deuterium exchange mass spectrometry to map binding interfaces between FBXW2 and its substrates.