FBXO28 Antibody

What applications are FBXO28 antibodies suitable for?

FBXO28 antibodies have been validated for multiple research applications based on extensive testing. According to available data, FBXO28 antibodies can be reliably used in:

These applications enable researchers to detect FBXO28 in various experimental contexts, from protein expression analysis to localization studies . When selecting an antibody for a specific application, researchers should consider the target species, as most available antibodies have been tested primarily on human samples with some cross-reactivity to mouse and rat models.

How should I validate the specificity of an FBXO28 antibody?

Validating antibody specificity is crucial for ensuring reliable experimental results. For FBXO28 antibodies, consider implementing the following validation strategy:

• Positive Control Testing: Use cell lines known to express FBXO28, such as HEK-293T, HeLa, or PC-3 cells .

• Molecular Weight Verification: Confirm detection at the expected molecular weight of approximately 42 kDa in Western blot applications .

• Knockdown/Knockout Validation: Compare antibody signal between wild-type cells and those where FBXO28 has been knocked down or knocked out using siRNA or CRISPR-Cas9 technologies. Effective knockdown should be confirmed using qRT-PCR and Western blot as demonstrated in functional assays .

• Signal Specificity: For IHC applications, compare staining patterns with literature references and include appropriate negative controls by omitting primary antibody. Antigen retrieval with TE buffer pH 9.0 is recommended for optimal results .

• Cross-Reactivity Assessment: If working with non-human samples, carefully evaluate cross-reactivity data, as most FBXO28 antibodies are optimized for human samples but may have reactivity with mouse and rat models .

These validation steps will help ensure that experimental observations are truly related to FBXO28 and not due to non-specific binding or cross-reactivity.

How does FBXO28 expression and activity correlate with cancer progression?

FBXO28 has demonstrated significant correlations with cancer progression in multiple studies. Research has revealed several important relationships:

• Expression Levels: FBXO28 is highly expressed in several cancer types compared to corresponding normal tissues. In pancreatic cancer (PC), elevated FBXO28 expression negatively correlates with patient survival prognosis .

• Proliferation Effects: Functional studies demonstrate that FBXO28 overexpression markedly increases proliferative capacity of cancer cells, while its downregulation hinders proliferation. This has been confirmed through CCK-8 and EdU tests, as well as colony formation assays .

• Cell Cycle Influence: FBXO28 upregulation increases expression of Cyclin E1, CDK2, and CDK4 while decreasing P27 expression. Flow cytometry studies show that FBXO28 upregulation promotes cancer cell conversion from G1 to S phase .

• Invasion and Metastasis: In vitro and in vivo experiments show that FBXO28a overexpression enhances migration and invasive capabilities of cancer cells. This is associated with increased N-cadherin and vimentin expression and decreased E-cadherin expression, indicating promotion of epithelial-mesenchymal transition .

• In Vivo Tumor Growth: In mouse models, tumors with FBXO28 overexpression showed significantly larger volumes and weights compared to control groups, with enhanced staining of proliferation markers Ki67 and PCNA .

These findings collectively suggest that FBXO28 promotes cancer progression through multiple mechanisms affecting cell proliferation, cell cycle regulation, and metastatic potential.

What is the relationship between FBXO28 phosphorylation and its function?

FBXO28 phosphorylation is a critical regulatory mechanism that directly impacts its functional activity:

Understanding the phosphorylation status of FBXO28 is therefore essential when studying its biological functions, as this post-translational modification directly influences its activity as an E3 ligase and its impact on downstream pathways.

How can I design experiments to study FBXO28-substrate interactions?

Designing experiments to study FBXO28-substrate interactions requires a multifaceted approach:

• Co-Immunoprecipitation (Co-IP):

  • Utilize antibodies against FBXO28 to pull down protein complexes from cell lysates.

  • Analyze precipitated proteins by mass spectrometry to identify potential interacting partners.

  • Confirm interactions by reverse Co-IP using antibodies against the identified target proteins.

  • This approach successfully identified SMARCC2 as a target of FBXO28 in pancreatic cancer research .

• In Vitro Ubiquitylation Assays:

  • Immunopurify wild-type and mutant (e.g., S344A, S344E) SCF^FBXO28 complexes.

  • Combine with purified potential substrate proteins, E1, E2 enzymes, ubiquitin, and ATP.

  • Detect ubiquitylation by Western blot analysis using substrate-specific antibodies.

  • This method successfully demonstrated the differential ubiquitylation activity of phosphorylated versus non-phosphorylated FBXO28 toward MYC .

• In Vivo Ubiquitylation Assays:

  • Co-express tagged versions of FBXO28 (wild-type or mutants), the potential substrate, and HA-tagged ubiquitin in cells.

  • Treat cells with proteasome inhibitors (e.g., MG132) to prevent degradation of ubiquitylated proteins.

  • Immunoprecipitate the substrate and detect ubiquitylation by Western blot using anti-HA antibodies.

  • Compare cells in different cell cycle phases (e.g., G1 versus S phase) to assess cell cycle-dependent effects .

• Protein Stability Assays:

  • Treat cells with cycloheximide to inhibit new protein synthesis.

  • Monitor substrate protein levels over time by Western blot in cells with normal, overexpressed, or depleted FBXO28.

  • This approach revealed that FBXO28 regulates SMARCC2 stability in pancreatic cancer cells .

These complementary approaches can comprehensively characterize the interactions between FBXO28 and its substrates, as well as the functional consequences of these interactions.

What methodologies can be used to study the non-proteolytic ubiquitylation mediated by FBXO28?

Studying non-proteolytic ubiquitylation mediated by FBXO28 requires specialized techniques that distinguish between degradative and non-degradative ubiquitin modifications:

• Ubiquitin Chain Topology Analysis:

  • Utilize ubiquitin mutants (e.g., K48R, K63R) to determine which lysine residues in ubiquitin are used for chain formation.

  • K48-linked chains typically signal for proteasomal degradation, while K63-linked chains often mediate non-proteolytic functions.

  • Employ mass spectrometry to analyze ubiquitin linkage types on FBXO28 substrates.

  • Research has shown that FBXO28 can direct non-proteolytic ubiquitylation of MYC, which affects transcriptional activity rather than protein stability .

• Proteasome Inhibition Studies:

  • Compare substrate levels and function with and without proteasome inhibitors (e.g., MG132, bortezomib).

  • For non-proteolytic ubiquitylation, substrate protein levels should remain stable even without proteasome inhibition, while functional changes may still occur.

  • This approach helps distinguish between degradative and non-degradative consequences of ubiquitylation.

• Chromatin Immunoprecipitation (ChIP):

  • For transcription factors like MYC that are subject to non-proteolytic ubiquitylation by FBXO28, perform ChIP to assess binding to target gene promoters.

  • Compare binding patterns and recruitment of cofactors (e.g., p300) in conditions of normal, enhanced, or reduced FBXO28 activity.

  • This method revealed that FBXO28-mediated ubiquitylation of MYC enhances recruitment of the cofactor p300 to MYC target gene promoters .

• Protein-Protein Interaction Dynamics:

  • Use fluorescence resonance energy transfer (FRET) or bioluminescence resonance energy transfer (BRET) to monitor real-time interactions between substrates and their binding partners.

  • Compare interaction dynamics in cells with normal, overexpressed, or depleted FBXO28 to assess how non-proteolytic ubiquitylation affects protein complex formation.

These methodologies provide complementary information about the non-proteolytic consequences of FBXO28-mediated ubiquitylation, which can include altered protein-protein interactions, subcellular localization, or enzymatic activities.

How can I establish models to study FBXO28's role in cancer progression?

Establishing robust models to study FBXO28's role in cancer progression requires a combination of in vitro and in vivo approaches:

• Cell Line Models with Modified FBXO28 Expression:

  • Generate stable cell lines with FBXO28 overexpression, knockdown, or knockout using lentiviral vectors or CRISPR-Cas9 technology.

  • Validate expression changes at both mRNA (qRT-PCR) and protein (Western blot) levels.

  • Research has successfully used this approach to study FBXO28's role in pancreatic cancer cell lines .

• Phosphorylation-Specific Mutants:

  • Create cell lines expressing phospho-deficient (S344A) or phospho-mimetic (S344E) FBXO28 mutants.

  • These models help distinguish between phosphorylation-dependent and -independent functions of FBXO28.

  • Studies have shown differential effects of these mutants on ubiquitin ligase activity toward MYC .

• In Vivo Xenograft Models:

  • Inject modified cancer cells subcutaneously into immunodeficient mice to assess tumor growth.

  • Measure tumor volume, weight, and perform immunohistochemical analysis for proliferation markers (Ki67, PCNA) and FBXO28 expression.

  • This approach revealed that FBXO28 overexpression significantly increases tumor growth in vivo .

• Metastasis Models:

  • Utilize tail vein injection or orthotopic implantation of modified cancer cells to assess metastatic potential.

  • For liver metastasis models, quantify the number and size of metastatic lesions by hematoxylin-and-eosin staining.

  • Research has shown that FBXO28 overexpression promotes liver metastasis in mouse models .

• Patient-Derived Xenografts (PDX):

  • Implant tumor tissue from patients directly into immunodeficient mice.

  • Analyze FBXO28 expression and phosphorylation in relation to tumor growth and response to therapies.

  • This model better preserves the heterogeneity and characteristics of human tumors.

These complementary models provide a comprehensive understanding of FBXO28's role in cancer progression, from molecular mechanisms to in vivo tumor growth and metastasis.

What are the optimal conditions for detecting FBXO28 phosphorylation in clinical samples?

Detecting FBXO28 phosphorylation in clinical samples presents several technical challenges that require specific methodological considerations:

These optimized conditions facilitate reliable detection of FBXO28 phosphorylation in clinical samples, enabling its evaluation as a potential biomarker for cancer prognosis and treatment response.

How can I design functional studies to elucidate FBXO28's role in transcriptional regulation?

Designing functional studies to investigate FBXO28's role in transcriptional regulation requires a comprehensive approach:

• Gene Expression Profiling:

  • Perform RNA-sequencing or microarray analysis in cells with FBXO28 knockdown, overexpression, or mutation.

  • Conduct gene set enrichment analysis (GSEA) to identify affected pathways.

  • Research has identified significantly enriched GO categories associated with FBXO28 knockdown in HCT116 cells, with 71% (1200/1690) of affected genes showing downregulation .

• Chromatin Immunoprecipitation (ChIP) Studies:

  • Perform ChIP-seq to identify genomic regions where FBXO28 or its substrate transcription factors (e.g., MYC) bind.

  • Compare binding patterns between wild-type cells and those with FBXO28 manipulation.

  • Include analysis of co-factors such as p300, as FBXO28-mediated ubiquitylation has been shown to affect co-factor recruitment .

• Sequential ChIP (Re-ChIP):

  • Use this technique to determine if FBXO28 and its substrate transcription factors simultaneously occupy the same genomic regions.

  • This approach can help establish direct versus indirect effects of FBXO28 on transcriptional regulation.

• Luciferase Reporter Assays:

  • Design luciferase constructs containing promoters of genes regulated by FBXO28-targeted transcription factors.

  • Compare reporter activity in cells with normal, overexpressed, or depleted FBXO28.

  • Include phosphorylation-site mutants (S344A, S344E) to assess the impact of FBXO28 phosphorylation on transcriptional activity.

• Integrative Analysis with Clinical Data:

  • Correlate expression of FBXO28 and its target genes in patient samples.

  • Research has found associations between FBXO28 expression and MYC target gene expression in human breast cancer, supporting the functional connection observed in laboratory studies .

• Transcription Factor Activity Assays:

  • Use systems like the Gal4-UAS system to directly measure transcription factor activity.

  • Assess how FBXO28 manipulation affects the activity of specific transcription factors independent of their expression levels.

These complementary approaches provide a comprehensive understanding of how FBXO28 influences transcriptional programs in normal and disease states, particularly through its interaction with transcription factors like MYC.

What are the critical controls needed for FBXO28 antibody-based experiments?

Implementing appropriate controls is essential for ensuring the validity and reproducibility of FBXO28 antibody-based experiments:

• Antibody Specificity Controls:

  • Positive Controls: Include lysates from cells known to express FBXO28 (e.g., HEK-293T, HeLa, PC-3) .

  • Negative Controls:

    • For Western blot: Include lysates from cells with FBXO28 knockdown or knockout.

    • For IHC/IF: Omit primary antibody while maintaining all other steps.

  • Peptide Competition: Pre-incubate antibody with excess immunizing peptide to block specific binding sites.

• Application-Specific Controls:

  • Western Blot:

    • Include molecular weight markers to confirm detection at the expected size (42 kDa) .

    • Use loading controls (e.g., GAPDH, β-actin) to normalize protein levels.

  • Immunohistochemistry:

    • Include positive and negative tissue controls in each staining batch.

    • For antigen retrieval optimization, test multiple conditions (e.g., TE buffer pH 9.0 versus citrate buffer pH 6.0) .

  • Immunoprecipitation:

    • Include IgG control to identify non-specific binding.

    • Perform reverse IP to confirm interactions.

• Experimental Validation Controls:

  • Expression Manipulation:

    • Compare results between wild-type, overexpression, and knockdown conditions.

    • Include rescue experiments with re-expression of siRNA-resistant FBXO28 to confirm specificity of knockdown effects.

  • Phosphorylation Studies:

    • Include phosphatase-treated samples as negative controls for phospho-specific antibodies.

    • Compare wild-type FBXO28 with phospho-mutants (S344A, S344E) .

• Technical Replicates and Reproducibility Controls:

  • Perform at least three independent biological replicates.

  • Include technical replicates within each experiment.

  • Ensure consistent antibody lots across experiments when possible.

These controls help ensure that observations attributed to FBXO28 are specific and reliable, reducing the risk of artifacts or misinterpretation of results.

How should I optimize protein extraction protocols for detecting FBXO28 and its post-translational modifications?

Optimizing protein extraction protocols is crucial for effectively detecting FBXO28 and its post-translational modifications, particularly phosphorylation:

• Cell/Tissue Lysis Buffer Composition:

  • Base Buffer: Use RIPA buffer (for general applications) or NP-40/Triton X-100 buffer (for co-immunoprecipitation).

  • Protease Inhibitors: Include a complete cocktail (e.g., PMSF, aprotinin, leupeptin, pepstatin A).

  • Phosphatase Inhibitors: Critical for preserving phosphorylation; include sodium fluoride (50 mM), sodium orthovanadate (1 mM), β-glycerophosphate (10 mM), and sodium pyrophosphate (5 mM).

  • Deubiquitinase Inhibitors: Add N-ethylmaleimide (10 mM) when studying ubiquitylation.

• Lysis Conditions:

  • Temperature: Perform all steps on ice to minimize protein degradation and dephosphorylation.

  • Sonication: Brief sonication (3-5 cycles of 10 seconds on/10 seconds off) can improve extraction of nuclear proteins like FBXO28.

  • Incubation Time: Limit to 30 minutes to minimize post-lysis modifications.

• Subcellular Fractionation:

  • Consider separating nuclear and cytoplasmic fractions when studying FBXO28, as it has nuclear functions related to transcriptional regulation .

  • Verify fraction purity using markers (e.g., HDAC1 for nuclear, GAPDH for cytoplasmic).

• Sample Processing for Phosphorylation Analysis:

  • Rapid Processing: Minimize time between sample collection and lysis.

  • Phospho-Enrichment: For low-abundance phospho-proteins, consider phospho-peptide enrichment techniques such as titanium dioxide (TiO2) or immobilized metal affinity chromatography (IMAC) prior to mass spectrometry analysis.

  • Cell Synchronization: For cell cycle-dependent phosphorylation, synchronize cells in specific phases (e.g., thymidine block for S phase, nocodazole for M phase).

• Sample Storage:

  • Short-term: Store samples at -80°C with phosphatase inhibitors.

  • Long-term: Add 5-10% glycerol to prevent freeze-thaw damage.

  • Avoid Repeated Freeze-Thaw: Aliquot samples to minimize degradation during repeated freezing and thawing.

Optimized extraction protocols ensure the preservation of FBXO28's native state and post-translational modifications, enabling more accurate analysis of its expression, interactions, and functional state in experimental and clinical samples.

What considerations are important when designing experiments to study FBXO28 in different cancer types?

When designing experiments to study FBXO28 in different cancer types, several important considerations should be addressed to ensure relevant and interpretable results:

• Baseline Expression Analysis:

  • Tissue-Specific Expression: Determine the baseline expression of FBXO28 in normal tissues corresponding to the cancer type of interest.

  • Cancer-Specific Alterations: Analyze cancer databases (e.g., TCGA, ICGC) to identify cancer types with FBXO28 alterations (expression changes, mutations, copy number variations).

  • Prognostic Significance: Assess whether FBXO28 expression or phosphorylation correlates with clinical outcomes in specific cancer types, as has been demonstrated for breast cancer and pancreatic cancer .

• Cell Line Selection:

  • Panel Diversity: Include multiple cell lines representing the cancer type's heterogeneity.

  • Molecular Subtypes: Ensure representation of different molecular subtypes within the cancer type.

  • FBXO28 Status: Select cell lines with varying levels of FBXO28 expression to observe differential effects.

  • Validated Models: Use well-characterized models such as HEK-293T, HeLa, and PC-3 cells, which have been validated for FBXO28 studies .

• Cancer-Specific Pathway Analysis:

  • Key Oncogenic Drivers: Investigate FBXO28's interaction with cancer-specific oncogenic drivers (e.g., MYC in breast cancer) .

  • Tissue-Specific Substrates: Identify potential tissue-specific substrates of FBXO28 (e.g., SMARCC2 in pancreatic cancer) .

  • Context-Dependent Functions: Assess whether FBXO28's function varies across cancer types due to different cellular contexts.

• In Vivo Model Selection:

  • Cancer Type Relevance: Choose models that accurately recapitulate the cancer type's biology.

  • Metastasis Assessment: For highly metastatic cancers, include appropriate metastasis models (e.g., liver metastasis model for pancreatic cancer) .

  • Microenvironment Consideration: Consider using orthotopic models that provide the appropriate tissue microenvironment.

• Translational Research Design:

  • Patient Sample Availability: Ensure access to sufficient patient samples for validation studies.

  • Treatment Correlation: Design studies to correlate FBXO28 status with response to standard-of-care treatments for the specific cancer type.

  • Biomarker Potential: Evaluate FBXO28 or phospho-FBXO28 as potential prognostic or predictive biomarkers in the context of the specific cancer type.

These considerations help ensure that experiments studying FBXO28 in different cancer types are biologically relevant, technically sound, and clinically informative, ultimately contributing to a better understanding of cancer-specific mechanisms and potential therapeutic strategies.

What emerging technologies could advance our understanding of FBXO28 function?

Several cutting-edge technologies offer promising opportunities to deepen our understanding of FBXO28 function:

• CRISPR-Based Screening Approaches:

  • CRISPR Activation/Inhibition: CRISPRa and CRISPRi systems can provide more nuanced modulation of FBXO28 expression compared to traditional overexpression or knockout approaches.

  • CRISPR Base Editing: Precise modification of specific nucleotides to introduce or correct phosphorylation site mutations (e.g., S344) without disrupting the entire gene.

  • CRISPR Screens: Genome-wide or targeted screens to identify synthetic lethal interactions with FBXO28 in cancer contexts.

• Advanced Proteomics Techniques:

  • Proximity Labeling: BioID or APEX2-based approaches to identify proteins in the vicinity of FBXO28 in living cells, potentially revealing novel interaction partners.

  • Crosslinking Mass Spectrometry: Identification of direct protein-protein interaction interfaces between FBXO28 and its substrates or complex components.

  • Targeted Proteomics: Development of multiple reaction monitoring (MRM) assays for precise quantification of FBXO28, its phosphorylated forms, and substrates across multiple samples.

• Live-Cell Imaging Technologies:

  • FRET/BRET Biosensors: Development of sensors to monitor FBXO28 activity, phosphorylation status, or substrate interactions in real-time.

  • Optogenetics: Light-controlled activation or inhibition of FBXO28 function to study temporal aspects of its activity.

  • Live-Cell Single-Molecule Tracking: Visualization of FBXO28 dynamics during cell cycle progression and in response to various stimuli.

• Structural Biology Approaches:

  • Cryo-EM: Determination of the structure of the SCF^FBXO28 complex, potentially in complex with substrates.

  • Hydrogen-Deuterium Exchange Mass Spectrometry: Investigation of conformational changes induced by phosphorylation or substrate binding.

  • AlphaFold2/RoseTTAFold: Computational prediction of FBXO28 structure and interaction interfaces to guide experimental design.

• Single-Cell Technologies:

  • Single-Cell Proteomics: Analysis of FBXO28 expression and phosphorylation heterogeneity within tumors.

  • Single-Cell Transcriptomics: Assessment of the impact of FBXO28 on gene expression programs at single-cell resolution.

  • Spatial Transcriptomics/Proteomics: Mapping of FBXO28 expression and activity within the tumor microenvironment.

These emerging technologies hold great promise for advancing our understanding of FBXO28 function, potentially revealing new aspects of its regulation, identifying novel substrates, and clarifying its role in cancer biology.

How might understanding FBXO28 biology contribute to cancer therapeutics?

Understanding FBXO28 biology presents several promising avenues for cancer therapeutic development:

• Direct Targeting of FBXO28:

  • Small Molecule Inhibitors: Development of compounds that disrupt FBXO28's interaction with specific substrates or with the SCF complex.

  • Protein Degradation Approaches: PROTAC (Proteolysis Targeting Chimera) technology could be employed to induce FBXO28 degradation in cancer cells.

  • Phosphorylation Inhibition: Given the importance of S344 phosphorylation for FBXO28 function , inhibitors that block this phosphorylation event could modulate its activity.

• CDK Inhibition as an Indirect Approach:

  • CDK1/2 Inhibitors: Since FBXO28 is activated by CDK1/2-mediated phosphorylation , CDK inhibitors might partially exert their anti-cancer effects through FBXO28 inhibition.

  • Cell Cycle Phase-Specific Therapies: Targeting cancer cells in S phase, when FBXO28 activity is highest, might provide a therapeutic window.

• Exploiting Synthetic Lethality:

  • Identifying Dependencies: Systematic screens for genes that, when inhibited, are selectively lethal to cells with high FBXO28 expression or activity.

  • Combining with Existing Therapies: Determining whether FBXO28 status affects sensitivity to standard chemotherapies, targeted agents, or immunotherapies.

• Biomarker Development:

  • Predictive Biomarkers: Development of FBXO28 or phospho-FBXO28 as biomarkers to predict response to therapies targeting related pathways (e.g., MYC inhibitors).

  • Prognostic Stratification: Using FBXO28 status to identify high-risk patients who might benefit from more aggressive treatment approaches.

  • Monitoring Treatment Response: Tracking changes in FBXO28 phosphorylation as a pharmacodynamic marker of CDK inhibitor efficacy.

• Targeting FBXO28 Substrates and Pathways:

  • MYC Pathway Inhibition: Since FBXO28 enhances MYC transcriptional activity , combining FBXO28 inhibition with MYC pathway inhibitors might have synergistic effects.

  • SMARCC2 Modulation: In pancreatic cancer, where FBXO28 targets SMARCC2 for degradation , strategies to stabilize SMARCC2 might counteract FBXO28's oncogenic effects.

These therapeutic strategies highlight the potential clinical significance of FBXO28 research, suggesting that a deeper understanding of its biology could lead to novel approaches for cancer treatment, particularly for aggressive cancers with poor prognosis like pancreatic cancer and certain subtypes of breast cancer.