FBXO25 Antibody

What is FBXO25 and why is it important to study?

FBXO25 is a member of the F-box protein family characterized by an approximately 40 amino acid F-box motif. These proteins constitute one of the four subunits of ubiquitin protein ligase complex called SCFs (SKP1-cullin-F-box), which function in phosphorylation-dependent ubiquitination . FBXO25 belongs to the Fbxs class of F-box proteins, which contain either different protein-protein interaction modules or no recognizable motifs beyond the F-box domain .

The importance of studying FBXO25 stems from its involvement in several biological processes. It forms a functional ubiquitin ligase complex with Skp1, Cul1, and Rbx1, and is concentrated in novel nuclear structures termed FBXO25-associated nuclear domains (FANDs) . Furthermore, FBXO25 gene variants have been linked to genetically inherited cerebral disorders, and its expression increases in response to interferon β treatment and viral infection, suggesting potential roles in inflammation and tumorigenesis .

What types of FBXO25 antibodies are available for research use?

Several types of FBXO25 antibodies are available for research applications:

• Polyclonal antibodies: These are commonly raised in rabbits and typically target specific regions of the FBXO25 protein, such as the N-terminal region . For example, there are polyclonal antibodies targeting synthetic peptides located within regions such as "LGEAFNRLDFSSAIQDIRRFNYVVKLLQLIAKSQLTSLSGVAQKNYFNIL" .

• Region-specific antibodies: Some antibodies are designed to target specific domains of FBXO25, such as the N-terminal region (residues 2-62), which has been used to generate effective antibodies for immunolocalization microscopy and immunoblot studies .

These antibodies are typically supplied in liquid format with preservatives such as sodium azide and stabilizers like sucrose, at concentrations around 0.5 mg/ml .

How do I select the appropriate FBXO25 antibody for my experimental design?

When selecting an FBXO25 antibody for your research, consider these methodological criteria:

• Application compatibility: Determine if the antibody has been validated for your intended application. For instance, while many FBXO25 antibodies are validated for Western blot (WB) analysis , verification for immunofluorescence, immunoprecipitation, or flow cytometry may vary among products.

• Species reactivity: Check both tested and predicted species reactivity to ensure compatibility with your experimental model. Some antibodies show confirmed reactivity with human FBXO25 and predicted reactivity across multiple species including mouse, rat, cow, dog, guinea pig, horse, pig, rabbit, and zebrafish based on sequence homology .

• Target region: Consider which region of FBXO25 your study focuses on. Antibodies targeting different epitopes may yield different results depending on protein conformation, post-translational modifications, or interaction with other proteins in your experimental conditions.

• Validation data: Review available validation data, including positive control samples used in testing, such as DU145, 293T cells, or mouse testis tissues that have been used to validate certain FBXO25 antibodies .

• Scientific literature: Cross-reference with publications that have successfully used specific FBXO25 antibodies for applications similar to yours.

How can I optimize immunofluorescence protocols for detecting FBXO25 in nuclear domains?

Optimizing immunofluorescence protocols for FBXO25 nuclear domain detection requires careful attention to several methodological aspects:

• Fixation method: For detecting nuclear proteins like FBXO25, 4% paraformaldehyde fixation is often effective. The search results indicate successful detection of FBXO25 in nuclear domains using standard formaldehyde fixation protocols .

• Antibody dilution and incubation: Based on published protocols, antibody incubations should be performed for 1 hour at room temperature in PBS/2% BSA. For FBXO25 detection, affinity-purified antibodies raised against specific fragments (such as residues 2-62) have been successfully used .

• Secondary antibody selection: Fluorescent secondary antibodies such as Alexa 488- and Alexa 594-coupled antibodies have been effectively used for visualizing FBXO25. These should be carefully selected to minimize cross-reactivity with other primary antibodies if performing co-localization studies .

• Controls: Include a preincubation control where the FBXO25 fragment is preincubated with affinity-purified antibodies to abolish signal, confirming antibody specificity .

• Imaging settings: For quantitative analysis of FBXO25-associated nuclear domains (FANDs), confocal microscopy has been successfully employed, with systematic counting of FANDs in randomly chosen fields (e.g., 100 cells from four independent microscope slides) .

• Co-localization studies: To examine associations with other nuclear structures, double-labeling with markers such as PML, SC35, SMN, p80-coilin, or B23-nucleophosmin has proven informative for distinguishing FANDs from other nuclear bodies .

What methods are effective for studying FBXO25's role in the ubiquitin-proteasome pathway?

To investigate FBXO25's function in the ubiquitin-proteasome pathway, researchers can implement several methodological approaches:

• SCF complex reconstitution assays: Since FBXO25 forms a functional ubiquitin ligase complex with Skp1, Cul1, and Rbx1, reconstituting this complex in vitro or in cell culture can help study its enzymatic activity. This typically involves co-expression of these components followed by co-immunoprecipitation to verify complex formation .

• Ubiquitination assays: To assess FBXO25-mediated ubiquitination of potential substrates, in vitro or cell-based ubiquitination assays can be performed. These typically include expressing tagged versions of FBXO25 (such as HA-FBXO25-FLAG), potential substrates, and ubiquitin, followed by immunoprecipitation and detection of ubiquitin chains .

• Proteasome inhibition studies: Treating cells with proteasome inhibitors like MG132 (5 μM for 12 hours has been successfully used) can help accumulate ubiquitinated substrates of FBXO25, facilitating their identification and characterization .

• FBXO25 domain mutation analysis: Creating F-box deletion mutants (ΔF) of FBXO25 can help determine which domains are essential for substrate recognition versus ubiquitin ligase activity. Gateway recombination systems have been effectively used to generate such constructs .

• Polyglutamine aggregation assays: FBXO25's role in preventing protein aggregation can be studied using cell-based assays with polyglutamine-containing huntingtin protein. Evidence suggests that FBXO25-dependent ubiquitin ligase activity can prevent aggregation of such proteins in the nucleus of HEK293 cells .

How can I investigate the relationship between FBXO25 and transcriptional regulation?

To study FBXO25's relationship with transcriptional regulation, consider these methodological approaches:

• Transcription inhibition experiments: Since FBXO25-containing nuclear structures are affected by transcriptional activity, treatments with transcription inhibitors provide valuable insights. Successful protocols have used actinomycin D (5 and 0.05 μg/ml for 2 hours), α-amanitin (100 μM for 3 hours), or DRB (50 μg/ml for 5 hours) .

• Heat-shock treatment: Heat shock drastically affects the nuclear organization of FBXO25-containing structures, indicating their dynamic nature and relationship with cellular stress responses. This can be used as an experimental paradigm to study FBXO25 dynamics .

• Co-localization with transcription factors: Immunofluorescence co-localization studies can reveal relationships between FBXO25 and specific transcription factors or transcriptional machinery components.

• Chromatin immunoprecipitation (ChIP): Though not explicitly mentioned in the search results, ChIP assays could be employed to determine if FBXO25 associates with specific genomic regions, either directly or as part of protein complexes.

• Reporter gene assays: These could help determine if FBXO25 influences the expression of specific genes by co-transfecting FBXO25 expression constructs with reporter plasmids containing promoters of interest.

• RNA-seq following FBXO25 manipulation: Comparing transcriptomes after FBXO25 overexpression, knockdown, or knockout can reveal genes whose expression is influenced by FBXO25 activity.

What are common issues with FBXO25 antibody specificity and how can they be addressed?

When working with FBXO25 antibodies, researchers may encounter specificity issues that can be addressed through proper controls and optimization:

• Cross-reactivity verification: Antibodies should be tested against samples known to express or lack FBXO25. The search results indicate that FBXO25 is expressed in most mouse tissues tested except striated muscle , making muscle tissue a potential negative control for specificity testing.

• Antibody validation controls: When using FBXO25 antibodies for immunostaining, preincubation of the antibody with the immunizing peptide/protein fragment should abolish all signals, confirming specificity. This approach has been successfully used with affinity-purified antibodies raised against the recombinant fragment spanning residues 2-62 of FBXO25 .

• Multiple antibody approach: Using antibodies targeting different epitopes of FBXO25 can help confirm the specificity of observed signals. Commercial antibodies targeting different regions (such as N-terminal vs. internal or C-terminal epitopes) are available .

• Knockout/knockdown controls: While not explicitly mentioned in the search results, using FBXO25 knockout or knockdown samples as negative controls provides definitive validation of antibody specificity.

• Species-specific considerations: Verify the sequence homology between your experimental species and the immunogen sequence. For example, some antibodies show predicted homology of 93% with mouse FBXO25 based on the immunogen sequence, which may affect binding efficiency .

• Storage and handling: For optimal performance, store antibodies according to manufacturer recommendations. Short-term storage at 2-8°C (up to 1 week) and long-term storage at -20°C in small aliquots to prevent freeze-thaw cycles are typically recommended .

How do I interpret co-localization data between FBXO25 and other nuclear proteins?

Interpreting co-localization data between FBXO25 and other nuclear proteins requires rigorous analysis and consideration of several factors:

• Quantitative analysis: For robust co-localization analysis, count the total number of FBXO25-associated nuclear domains (FANDs), other nuclear structures of interest (e.g., clastosomes), and the number of colocalizing dots in a sufficient number of cells (e.g., 100 cells from randomly chosen fields across multiple independent slides) .

• Resolution limitations: Consider the resolution limits of your imaging system. Standard confocal microscopy has a resolution limit of approximately 200-250 nm, meaning structures appearing to co-localize might actually be separated but below the resolution limit.

• Z-stack analysis: Perform z-stack imaging to ensure that apparent co-localization in a single plane represents true three-dimensional co-localization rather than overlapping structures at different depths.

• Pearson's correlation coefficient: Calculate Pearson's correlation coefficient or other co-localization statistics to quantify the degree of co-localization between FBXO25 and other proteins.

• Biological significance: When interpreting co-localization between FBXO25 and other proteins, consider the biological context. Research has shown that FBXO25 nuclear domains contain high concentrations of ubiquitin conjugates and at least two other components of the ubiquitin-proteasome system: 20S proteasome and Skp1 . This suggests functional relationships rather than coincidental co-localization.

• Dynamic co-localization: Consider that co-localization may be dynamic and condition-dependent. For example, inhibition of transcription by actinomycin D or heat-shock treatment drastically affects the nuclear organization of FBXO25-containing structures .

What are the key considerations when comparing FBXO25 expression across different tissues or experimental conditions?

When comparing FBXO25 expression across different tissues or experimental conditions, researchers should consider these methodological aspects:

• Tissue-specific expression patterns: FBXO25 shows tissue-specific expression patterns, with predominant expression in the central nervous system and significant levels in intestine and kidney . Northern blot and RT-PCR studies have confirmed this expression pattern . Therefore, expression comparisons should account for this natural variation.

• Appropriate loading controls: When performing Western blots, use appropriate loading controls for the tissue or cell type being studied. Common loading controls used in FBXO25 studies include β-actin (1:3000) and β-tubulin (1:2000) .

• Antibody sensitivity: Different antibodies may have different sensitivities, potentially affecting the detection threshold across samples. Using the same antibody lot for all comparisons is recommended for consistency.

• Regulatory influences: FBXO25 expression can be influenced by factors such as interferon β treatment and virus infection . When comparing expression under different conditions, consider these potential regulatory influences.

• Quantification methods: For accurate comparison of FBXO25 levels, use quantitative methods such as densitometry for Western blots, quantitative PCR for mRNA levels, or quantitative image analysis for immunofluorescence data.

• Subcellular localization: FBXO25 shows specific subcellular localization patterns, particularly in nuclear domains. When comparing expression, consider not just total protein levels but also potential differences in subcellular distribution.

How can FBXO25 antibodies be used to study neurodegenerative disease mechanisms?

FBXO25 antibodies offer valuable tools for investigating neurodegenerative disease mechanisms, particularly given its high expression in the central nervous system and potential role in protein quality control:

• Polyglutamine disease models: FBXO25 has been implicated in preventing aggregation of polyglutamine-containing huntingtin protein in the nucleus . Researchers can use FBXO25 antibodies to study its co-localization with and potential regulation of polyglutamine-containing proteins in models of Huntington's disease and other polyglutamine expansion disorders.

• Cerebral disorder studies: Given that an FBXO25 gene variant has been linked to a genetically inherited cerebral disorder , antibodies can be used to compare expression and localization patterns between wild-type and disease-associated variants in patient samples or model systems.

• Stress-induced responses: Since heat shock affects FBXO25 nuclear structures , antibodies can help investigate how cellular stress responses in neurodegenerative conditions impact FBXO25 function and localization.

• Protein aggregation studies: In neurodegenerative diseases characterized by protein aggregation, FBXO25 antibodies can help determine if FBXO25-containing nuclear domains co-localize with or are altered by disease-associated protein aggregates.

• Therapeutic target validation: If FBXO25 is being investigated as a therapeutic target, antibodies can be used to validate target engagement by potential drugs or to monitor changes in FBXO25 expression or localization in response to treatment.

• Patient sample analysis: In post-mortem brain tissue from patients with neurodegenerative diseases, FBXO25 antibodies can be used to assess potential alterations in expression or localization compared to healthy controls.

What protocols are recommended for studying FBXO25's role in response to cellular stress?

To investigate FBXO25's role in cellular stress responses, consider these methodological approaches:

• Heat shock protocols: Heat shock treatment has been shown to affect FBXO25 nuclear organization drastically . Standard protocols typically involve exposing cells to 42-45°C for 30-60 minutes, followed by recovery at 37°C for various time points.

• Transcriptional inhibition: Use transcription inhibitors to study FBXO25 dynamics during transcriptional stress. Established protocols include:

  • Actinomycin D: 5 and 0.05 μg/ml for 2 hours

  • α-amanitin: 100 μM for 3 hours

  • DRB (dichlororibofuranosylbenzimidazole): 50 μg/ml for 5 hours

• Proteasome inhibition: Treat cells with the proteasome inhibitor MG132 (5 μM for 12 hours) to study how accumulation of ubiquitinated proteins affects FBXO25 localization and function .

• Interferon treatment: Since FBXO25 mRNA levels increase in response to interferon β treatment , treating cells with interferon β and monitoring FBXO25 expression and localization over time can provide insights into its role in immune responses.

• Viral infection models: FBXO25 expression changes have been observed following virus infection . Infecting cells with various viruses and monitoring FBXO25 dynamics can help elucidate its role in antiviral responses.

• Time-course imaging: Perform live-cell imaging with fluorescently tagged FBXO25 or fixed-cell immunofluorescence at various time points after stress induction to capture dynamic changes in FBXO25 localization and interactions.

How can I design experiments to identify novel substrates of FBXO25-mediated ubiquitination?

Identifying novel substrates of FBXO25-mediated ubiquitination requires a strategic experimental approach:

• Proteomic screening: Perform immunoprecipitation of FBXO25 followed by mass spectrometry to identify interacting proteins that may be potential substrates. Compare results between wild-type FBXO25 and F-box deletion mutants (ΔF) to distinguish between substrates and other interacting proteins .

• Ubiquitination assays: For candidate substrates, perform in vitro or cell-based ubiquitination assays. Express HA-FBXO25-FLAG (wild type and ΔF mutant) together with the candidate substrate and ubiquitin, followed by immunoprecipitation and detection of ubiquitinated species .

• Protein stability assays: Compare the stability of candidate substrates in cells expressing FBXO25 versus FBXO25-depleted cells. This can be done using cycloheximide chase assays to monitor protein degradation rates.

• Correlation with FBXO25 nuclear domains: Investigate whether candidate substrates co-localize with FBXO25-associated nuclear domains (FANDs), which contain high concentrations of ubiquitin conjugates .

• Validation in disease models: Given FBXO25's potential role in cerebral disorders and inflammation , validate candidate substrates in relevant disease models to assess physiological relevance.

• Domain mapping: For confirmed substrates, identify the specific domains or motifs required for FBXO25 recognition, typically through deletion or point mutation analysis of the substrate.

• Phosphorylation dependency: Since SCF complexes often recognize phosphorylated substrates, investigate whether phosphorylation of candidate substrates enhances their recognition by FBXO25.

What experimental approaches are recommended for studying FBXO25's influence on gene expression?

To investigate FBXO25's influence on gene expression, consider these methodological approaches:

• Transcriptome analysis: Perform RNA-seq or microarray analysis comparing gene expression profiles between control cells and cells with FBXO25 overexpression, knockdown, or knockout to identify genes regulated by FBXO25.

• ChIP-seq analysis: While not explicitly mentioned in the search results, chromatin immunoprecipitation followed by sequencing (ChIP-seq) using FBXO25 antibodies could identify genomic regions where FBXO25 associates, potentially affecting gene expression.

• Reporter gene assays: Construct reporter plasmids containing promoters of interest and measure reporter activity in the presence or absence of FBXO25 to assess direct effects on specific genes.

• Analysis of transcription factor stability: Since FBXO25 is part of an SCF ubiquitin ligase complex , investigate whether it regulates the stability of specific transcription factors, thereby indirectly influencing gene expression.

• Co-immunoprecipitation with transcriptional machinery: Perform co-immunoprecipitation experiments to identify interactions between FBXO25 and components of the transcriptional machinery.

• Dynamic analysis during transcriptional inhibition: Given that FBXO25 nuclear organization is affected by transcription inhibitors , study the dynamics of FBXO25 localization and interactions during transcriptional recovery after inhibitor removal.

• Correlation with cellular conditions: Analyze how FBXO25's influence on gene expression varies under different cellular conditions, such as during interferon β treatment or viral infection, conditions known to affect FBXO25 levels .

How might FBXO25 be involved in inflammation and immune responses?

Research indicates potential roles for FBXO25 in inflammation and immune responses, which can be investigated through these approaches:

• Interferon response pathway: Since FBXO25 mRNA levels increase in response to interferon β treatment , investigate its role in interferon signaling pathways. This could involve measuring interferon-stimulated gene expression in FBXO25-depleted cells compared to controls.

• Viral infection models: FBXO25 expression changes have been observed following virus infection . Study how FBXO25 affects viral replication, interferon production, and inflammatory cytokine expression in infected cells.

• Inflammatory signaling pathways: Investigate whether FBXO25 regulates key components of inflammatory signaling pathways such as NF-κB, which often relies on ubiquitination for activation or inhibition.

• Immune cell function: Examine FBXO25 expression and function in various immune cell types during activation, differentiation, and effector functions.

• Mouse models: Generate tissue-specific FBXO25 knockout mice, particularly in immune cells, to study its role in immune responses in vivo.

• Inflammatory disease correlation: Analyze FBXO25 expression in samples from patients with inflammatory diseases compared to healthy controls to identify potential correlations with disease status.