FBL19 Antibody
Description
Definition and Mechanism of Action
FBXL19 is a substrate-recognition component of the SCF (SKP1-CUL1-F-box protein) complex, mediating ubiquitination and degradation of target proteins. Key functions include:
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RHOA and RAC1 degradation: Regulates cell migration and cytoskeletal reorganization by targeting these GTPases for proteasomal degradation .
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IL1RL1 ubiquitination: Blocks IL-33-induced apoptosis by degrading its receptor .
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Chromatin remodeling: Recruits CDK-mediator to silent developmental genes and facilitates histone H2B mono-ubiquitination via RNF20 recruitment .
FBL19 antibodies enable detection and functional studies of FBXL19 in various experimental systems.
Antibody Types and Characteristics
FBL19 antibodies are categorized by their production methods and epitope recognition:
Monoclonal Antibodies
Polyclonal Antibodies
Notes:
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Monoclonal antibodies (e.g., Abcam’s EPR11957) offer higher specificity and reproducibility due to recombinant production .
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Polyclonal antibodies (e.g., Proteintech, Thermo Fisher) provide broader epitope coverage but may require optimization for sensitivity .
Western Blot (WB)
| Antibody | Dilution | Sample Type | Observed Bands | Reference |
|---|---|---|---|---|
| Abcam (ab172961) | 1/1000 | HT1080, A549, Mouse testis | ~76 kDa | |
| Proteintech (23624-1-AP) | 1:1000–1:4000 | HEK-293, HeLa | 60–75 kDa |
Immunocytochemistry/Immunofluorescence (ICC/IF)
Biological and Clinical Relevance
FBXL19 antibodies are pivotal in studying:
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Cancer biology: FBXL19 regulates TGFB1-induced E-cadherin downregulation and cell migration, linking it to epithelial-mesenchymal transition (EMT) .
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Inflammation: Mediates IL-33 signaling by degrading IL1RL1, modulating apoptosis and immune responses .
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Epigenetics: Facilitates chromatin remodeling by recruiting CDK8 to developmental gene promoters, influencing cell differentiation .
Challenges and Considerations
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Cross-reactivity: Polyclonal antibodies may show species cross-reactivity (e.g., Thermo Fisher’s antibody predicts rat reactivity) .
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Band variability: Observed molecular weights (60–75 kDa) may differ from predicted (76 kDa) due to post-translational modifications or proteolytic cleavage .
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Experimental optimization: Dilution ranges for WB (1:1000–1:4000) and ICC/IF (1–4 µg/mL) require titration .
Research Trends and Future Directions
Product Specs
Q&A
Here’s a structured collection of FAQs tailored for researchers working with FBL19 (FBXL19) antibodies, focusing on academic research scenarios. The questions are categorized into basic and advanced tiers, with methodological guidance and data-driven insights derived from the provided sources.
How do I determine the appropriate applications for FBXL19 antibodies in experimental models?
FBXL19 antibodies are primarily used to study its role as an E3 ubiquitin ligase component and chromatin regulator. Key applications include:
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Western blot (WB): Validate FBXL19 overexpression or knockdown in cell lysates (e.g., HEK293T transfected systems) using antibodies like ab172961 .
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Immunocytochemistry (ICC): Localize FBXL19 in subcellular compartments (e.g., nuclear vs. cytoplasmic distribution) .
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Functional assays: Investigate FBXL19-mediated degradation of substrates like RHOA or RAC1 via co-immunoprecipitation and ubiquitination assays .
Validation Tip: Use overexpression lysates (e.g., LS054620) as positive controls and siRNA knockdown to confirm target specificity .
What methods ensure antibody specificity for FBXL19 in heterogeneous samples?
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Knockdown/knockout controls: Compare signal intensity in FBXL19-silenced vs. wild-type cells.
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Competition assays: Pre-incubate the antibody with recombinant FBXL19 protein to block binding.
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Cross-reactivity checks: Test against related F-box proteins (e.g., FBXW7) using lysates from tissues with high homologous expression .
Example Data:
| Method | Result | Source |
|---|---|---|
| WB (HEK293T lysates) | 75.7 kDa band in transfected cells, absent in untransfected | |
| ICC (HT1080 cells) | Nuclear and cytoplasmic staining, abolished by siRNA |
What are the most well-characterized substrates or pathways involving FBXL19?
FBXL19 regulates:
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Cytoskeletal dynamics: Degrades RAC1/RAC3 to inhibit TGFB1-induced cell migration .
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Nuclear function: Binds unmethylated CpG DNA and recruits CDK8/RNF20 for histone H2B ubiquitination .
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Immune modulation: Targets IL1RL1 for degradation, blocking IL-33-mediated apoptosis .
Experimental Design: Use SPR (surface plasmon resonance) to quantify binding kinetics between FBXL19 and substrates like RAC1 .
How can I resolve contradictory data on FBXL19’s role in RHOA vs. RAC1 degradation across cell types?
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Context-dependent analysis: FBXL19 activity may vary with ERK2 activation status or cellular stress. Design experiments comparing:
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ERK2-inhibited vs. active conditions.
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Cancer vs. non-transformed cell lines.
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Multi-omics integration: Couple proteomics (substrate turnover rates) with transcriptomics to identify upstream regulators .
Key Finding: FBXL19-mediated RHOA degradation is ERK2-dependent, while RAC1 degradation is TGFB1-induced .
What strategies optimize FBXL19 antibody use in chromatin interaction studies?
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Chromatin fractionation: Separate nuclear/cytoplasmic fractions to assess FBXL19-DNA binding.
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CUT&Tag sequencing: Map FBXL19-bound genomic regions using antibody-compatible protocols.
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CRISPR-dCas9 fusion: Tag FBXL19 with dCas9 to recruit it to specific loci and monitor histone modifications .
Data Insight: FBXL19 recruits RNF20 to promote H2B ubiquitination at developmental gene promoters .
How do I design in vivo models to study FBXL19’s therapeutic potential?
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Transgenic models: Generate tissue-specific FBXL19 knockouts to study metastasis or immune dysregulation.
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Xenograft studies: Implant FBXL19-overexpressing cancer cells and monitor tumor progression via bioluminescence.
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Pharmacokinetics (PK): Adapt population PK models (e.g., two-compartment systems) from murine antibody studies to predict FBXL19 antibody behavior .
PK Parameters from Analogous Studies:
Methodological Best Practices
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Antibody validation: Always include overexpression lysates and isotype controls.
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Data contradiction resolution: Perform dose-response assays to identify threshold effects (e.g., FBXL19’s dual role in pro-/anti-migratory signals) .
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Collaborative verification: Share antibody lots with independent labs to confirm reproducibility, especially for chromatin studies .
