FBX15 Antibody
Description
Introduction to FBX15 Antibody
FBX15 Antibody is a specialized immunological reagent designed to detect and study F-box protein 15 (FBX15), a substrate receptor component of the SCF-type E3 ubiquitin ligase complex. This antibody is critical for research into cellular processes such as protein ubiquitination, pluripotency, stress response, and pathogenicity in fungi. It is available in polyclonal forms (e.g., rabbit-derived) and conjugated variants (e.g., Alexa Fluor® 594) for diverse applications like Western blotting (WB), immunohistochemistry (IHC), and ELISA .
Applications in Research
FBX15 Antibody is utilized in multiple experimental contexts, with optimized protocols for specific techniques:
Key features include:
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Cross-reactivity: Detects human and mouse FBX15 with partial cross-reactivity to recombinant mouse FBX15 (~10%) .
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Epitope specificity: Targets regions such as aa 298–434 or isoform-specific domains .
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Observed molecular weight: ~55 kDa (vs. calculated 49 kDa), likely due to post-translational modifications .
Role in Embryonic Stem Cells
FBX15 is expressed in undifferentiated embryonic stem cells (ESCs) and early embryos, regulated by pluripotency factors Oct3/4 and Sox2 . While FBX15-null ESCs exhibit normal morphology and differentiation, its absence causes subfertility in mice due to mitochondrial dysfunction in germ cells .
Fungal Pathogenicity
In Aspergillus fumigatus, Fbx15 (homolog of FBX15) regulates stress response and virulence:
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Oxidative stress induction: Upregulates Fbx15 expression, enabling gliotoxin production and infection in mice .
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Dual function: Acts as part of SCF ligases and controls nuclear localization of the SsnF/Ssn6 co-repressor complex .
Cancer and Metabolic Research
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Lipid metabolism: FBX15 interacts with SKP1-Cullin-1-FBXO11 complexes to modulate heterochromatin protein A2/B1 ubiquitination in hepatocellular carcinoma .
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Immunoparalysis: Oroxylin A degrades CHOP via FBX15-mediated pathways in sepsis models .
Challenges and Considerations
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Titration requirements: Optimal dilutions vary by application and sample type .
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Cross-reactivity: Partial reactivity with mouse FBX15 necessitates species-specific validation .
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Biological redundancy: FBX15 is dispensable for ESC self-renewal and embryonic development, complicating functional studies .
Future Directions
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Fungal drug development: Targeting Fbx15 in Aspergillus fumigatus may inhibit stress adaptation and virulence .
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Stem cell reprogramming: FBX15 serves as a marker for induced pluripotency, enabling monitoring of reprogramming efficiency .
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Cancer therapeutics: Investigating FBX15’s role in lipid metabolism and immunoparalysis could uncover novel therapeutic targets .
Product Specs
Constituents: 50% Glycerol, 0.01M Phosphate Buffered Saline (PBS), pH 7.4
Q&A
What is the role of FBX15 in cellular stress responses?
FBX15 is an F-box protein that functions as part of SCF E3 ubiquitin ligase complexes. It plays a critical role in oxidative stress responses by regulating transcriptional repression and gliotoxin biosynthesis in Aspergillus fumigatus. Its activity is modulated by phosphorylation, which determines its localization in the nucleus or cytoplasm. For example, under oxidative stress, FBX15 levels increase rapidly, facilitating adaptive responses .
How can FBX15 expression be quantified during stress conditions?
FBX15 expression can be measured using real-time PCR (RT-PCR) for transcript levels and Western blotting for protein quantification. In A. fumigatus, FBX15 transcript levels show a peak at 40 minutes after oxidative stress induction, followed by a decline to basal levels. Protein levels mirror this pattern with a delay of approximately 20 minutes .
What experimental systems are suitable for studying FBX15 function?
FBX15 function can be studied using knockout models (e.g., ΔFBX15 mutants) or complementation assays in fungal strains like A. fumigatus. These systems allow researchers to assess changes in stress response, gliotoxin production, and virulence .
How does FBX15 regulate gliotoxin biosynthesis at the molecular level?
FBX15 interacts with SsnF/Ssn6, a transcriptional co-repressor complex, to repress gliotoxin biosynthesis genes under normal conditions. In ΔFBX15 mutants, the expression of genes such as gliZ and gliP increases significantly, leading to elevated gliotoxin production. This regulation is crucial for fungal virulence and adaptation to host environments .
What techniques can be used to study post-translational modifications of FBX15?
Post-translational modifications like phosphorylation can be studied using immunoblotting with specific antibodies against phosphorylated proteins or mass spectrometry-based proteomics. These techniques help elucidate how phosphorylation affects FBX15's localization and function during stress conditions .
How can contradictory data regarding FBX15's role in pluripotency be resolved?
Studies have shown that FBX15 is dispensable for embryonic stem cell self-renewal despite being a target of Oct3/4. Resolving contradictions requires comparative analysis of experimental models (e.g., mouse vs human systems), gene-targeting approaches, and functional assays like teratoma formation tests .
What are the implications of FBX15 deletion on fungal virulence?
In murine models of invasive pulmonary aspergillosis, ΔFBX15 mutants lose virulence entirely due to impaired stress adaptation mechanisms. Histopathological analyses show reduced fungal growth and immune infiltration compared to wild-type strains, highlighting FBX15's essential role in pathogenicity .
How can antibody specificity for FBX15 isoforms be validated?
Validation involves testing antibodies across multiple applications (e.g., Western blotting, ELISA) using positive controls like human testis tissue or recombinant proteins representing different isoforms of FBX15. Specificity can be confirmed by detecting distinct bands corresponding to expected molecular weights .
What controls should be included in experiments using FBX15 antibodies?
Controls should include:
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Negative controls: Samples lacking FBX15 expression (e.g., knockout models).
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Positive controls: Known FBX15-expressing tissues or recombinant proteins.
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Loading controls: Housekeeping proteins like GAPDH or β-actin for normalization in Western blot experiments .
How can researchers optimize antibody dilutions for different applications?
Antibody dilutions should be optimized experimentally based on application-specific recommendations:
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Western Blot: Typically 1:500–1:3000.
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Immunohistochemistry: Typically 1:200–1:800.
Titration experiments are essential to achieve optimal signal-to-noise ratios .
