FBXL17 Antibody

What is FBXL17 and what is its primary function in cellular systems?

FBXL17, also known as F-box/LRR-repeat protein 17, functions as a substrate-recognition component of the SCF (SKP1-CUL1-F-box protein) E3 ubiquitin ligase complex. This complex plays a critical role in protein quality control pathways, particularly in ensuring functional dimerization of BTB domain-containing proteins through a process known as dimerization quality control (DQC). FBXL17 specifically recognizes and binds to a conserved degron present at the interface of aberrant BTB dimers, facilitating their ubiquitination and subsequent degradation by the proteasome .

The SCF(FBXL17) complex mediates the ubiquitination and degradation of several key regulatory proteins. These include BACH1, SUFU (a negative regulator of Hedgehog signaling), and PRMT1 . By targeting SUFU for degradation, FBXL17 enables the release of GLI1 from SUFU-mediated inhibition, allowing proper Hedgehog signaling transduction . This regulatory mechanism is particularly significant in developmental processes and certain pathological conditions, including cancer.

FBXL17 also plays a critical role in neuronal function through its interaction with SPAST-M1 (Spastin), where it regulates microtubule dynamics that are essential for proper neuronal development and function. Disruption of this relationship has been implicated in hereditary spastic paraplegia (HSP), highlighting FBXL17's importance in neurological health .

What applications are FBXL17 antibodies most suitable for in research settings?

FBXL17 antibodies have been validated and proven effective for several key research applications, making them versatile tools for investigating this protein's function and interactions:

Western Blotting (WB) represents a primary application where FBXL17 antibodies demonstrate strong utility. This technique allows researchers to detect and quantify FBXL17 expression in cell or tissue lysates, enabling studies of expression changes under different experimental conditions. The rabbit polyclonal antibodies available commercially have been specifically validated for this application with human samples .

Immunohistochemistry on paraffin-embedded sections (IHC-P) offers another valuable application. This technique facilitates visualization of FBXL17 expression patterns in tissue sections, which can be particularly informative when comparing normal versus diseased states. The spatial distribution information obtained through IHC-P complements the quantitative data from Western blotting .

Immunoprecipitation (IP) represents a critical application for studying FBXL17's protein-protein interactions. FBXL17 antibodies have been successfully used to isolate the protein along with its binding partners from cell lysates. This approach has been instrumental in discovering and confirming interactions with proteins such as Sufu, BACH1, and Spastin . When implementing IP protocols, researchers should consider using proteasome inhibitors like MLN4924 to stabilize these interactions, as they may be transient due to the rapid degradation of FBXL17 targets .

For subcellular localization studies, immunofluorescence microscopy using FBXL17 antibodies can provide valuable insights into the protein's distribution within cells. This is particularly relevant given evidence of compartment-specific functions, such as nuclear regulation of SPAST-M1 .

How should researchers validate FBXL17 antibody specificity for their experimental systems?

Validating FBXL17 antibody specificity requires a multi-faceted approach to ensure reliable experimental results. A primary validation strategy involves siRNA/shRNA knockdown experiments, where researchers should observe a significant reduction in antibody signal following FBXL17 depletion. This approach has been successfully employed in studies examining FBXL17's interaction with Sufu and SPAST-M1 . To strengthen this validation, researchers should use at least two different siRNA sequences targeting distinct regions of FBXL17 mRNA to exclude off-target effects.

Overexpression controls provide another essential validation method. Researchers should compare the endogenous antibody signal to that of exogenously expressed FBXL17, ensuring the antibody recognizes both forms at appropriate molecular weights. In published studies validating FBXL17-Sufu interaction, researchers compared antibody detection of endogenous FBXL17 to an exogenously expressed FBXL17, confirming antibody specificity .

For more rigorous validation, peptide competition assays can be performed. By pre-incubating the antibody with its immunizing peptide/antigen and applying this mixture to samples in parallel with untreated antibody, researchers should observe diminished or eliminated signal in the peptide-treated condition if the antibody is specific.

The use of multiple antibodies targeting different epitopes of FBXL17 represents a gold-standard approach to validation. Concordant results with different antibodies strongly support specificity of detection. The Human Protein Atlas employs this approach for enhanced validation of antibodies .

Immunoprecipitation followed by mass spectrometry analysis can further confirm antibody specificity by verifying that FBXL17 is among the most abundant proteins in the precipitate. This technique has been successfully used to identify FBXL17 interaction partners in previous studies .

When available, testing the antibody in FBXL17 knockout or transgenic models provides the most definitive evidence of specificity. Complete absence of signal in knockout models would strongly support antibody specificity.

What is the role of FBXL17 in Hedgehog signaling pathway regulation?

FBXL17 serves as a critical regulator of the Hedgehog (Hh) signaling pathway through its targeted degradation of Suppressor of Fused (Sufu), a key negative regulator of this pathway. The molecular mechanism involves FBXL17 forming a functional SCF E3 ubiquitin ligase complex that specifically recognizes Sufu and targets it for ubiquitination in the nuclear compartment upon Hedgehog pathway activation. The subsequent proteasomal degradation of Sufu releases the Gli1 transcription factor from Sufu-mediated inhibition, allowing Gli1 to activate Hedgehog target gene expression .

This regulatory mechanism has been substantiated through multiple experimental approaches. Immunoprecipitation studies have demonstrated that FBXL17 specifically interacts with Sufu, while other F-box proteins tested do not show this interaction. The endogenous interaction between FBXL17 and Sufu has been validated in multiple cell lines including PC3 (prostate cancer) and DAOY (medulloblastoma) .

Manipulation of FBXL17 levels directly impacts Sufu protein abundance but not its mRNA levels, confirming post-translational regulation. FBXL17 depletion using siRNA increases Sufu protein levels, while FBXL17 overexpression decreases Sufu protein levels. Half-life measurements reveal that Sufu protein stability increases from approximately 6 hours to more than 12 hours in FBXL17-depleted cells, providing direct evidence for FBXL17's role in regulating Sufu turnover .

The functional consequences of this regulation on Hedgehog signaling are significant. FBXL17 silencing results in decreased Gli1 mRNA levels (a key Hh pathway target gene), while FBXL17 overexpression leads to increased Gli1 mRNA expression. These effects correlate directly with changes in Sufu protein levels, confirming the functional impact on Hh pathway activity .

In Hedgehog-dependent cancer cells, FBXL17 depletion impairs cell proliferation. Importantly, this proliferation defect can be rescued either by re-introduction of FBXL17 or by simultaneous knockdown of Sufu, confirming that the anti-proliferative effect of FBXL17 depletion is specifically mediated through Sufu accumulation .

How does the FBXL17-Spastin axis contribute to neurological disease models?

The FBXL17-Spastin axis represents a novel regulatory pathway with significant implications for neurological disorders, particularly hereditary spastic paraplegia (HSP). At the molecular level, FBXL17 forms an SCF E3 ubiquitin ligase complex that specifically targets SPAST-M1 (Spastin) for ubiquitination and degradation. This interaction occurs specifically through the BTB domain at the N-terminus of SPAST-M1. Protein chip analysis has demonstrated an inverse correlation between FBXL17 and SPAST-M1 protein levels both in vitro and during embryonic development in vivo .

The regulation mechanism involves compartment-specific processing. SPAST phosphorylation occurs exclusively in the cytoplasmic fraction through Casein Kinase 2 (CK2) activity, and this phosphorylation is involved in the poly-ubiquitination process. The SCF FBXL17 E3 ubiquitin ligase complex then degrades SPAST-M1 specifically in the nuclear fraction in a proteasome-dependent manner .

This regulatory pathway has profound implications for hereditary spastic paraplegia. The SPAST Y52C mutant, which contains an abnormality in the BTB domain, cannot interact with FBXL17, thereby escaping regulation by the SCF FBXL17 E3 ubiquitin ligase complex. This results in a loss of functionality with aberrant protein quantity. In neuronal models, this mutation leads to shortened axonal outgrowth, reduced proliferation rates, and poor differentiation capacity in 3D models .

The therapeutic potential of targeting this axis is particularly noteworthy. Inhibition of the SCF FBXL17 E3 ubiquitin ligase (using small chemical inhibitors or FBXL17 shRNA) decreases proteasome-dependent degradation of SPAST-M1 and induces axonal extension in normal neurons. More remarkably, inhibiting the SCF FBXL17 E3 ubiquitin ligase can rescue the phenotypic defects caused by the SPAST Y52C mutation . This suggests that targeting FBXL17 could provide a novel therapeutic approach for certain forms of HSP where spastin regulation is disrupted.

What experimental approaches can effectively study FBXL17-mediated protein degradation kinetics?

Investigating FBXL17-mediated protein degradation kinetics requires specialized techniques that capture the dynamic nature of protein ubiquitination and subsequent degradation. The cycloheximide (CHX) chase assay represents a fundamental approach, where new protein synthesis is blocked using cycloheximide and samples are collected at multiple time points thereafter. Western blotting analysis of target protein decay (such as Sufu or SPAST-M1) can then reveal degradation rates. Studies have shown that Sufu half-life increases from approximately 6 hours to more than 12 hours in FBXL17-depleted cells, providing quantitative measures of FBXL17's impact on degradation kinetics .

Ubiquitination assays provide direct visualization of FBXL17's enzymatic function. These typically involve co-transfection of tagged ubiquitin (e.g., HA-ubiquitin) with FBXL17 and its substrate, followed by immunoprecipitation of the substrate and detection of ubiquitin modifications via Western blot. This approach can determine both the extent and pattern of ubiquitination (e.g., K48 vs. K63 linkages) on FBXL17 targets.

Proteasome inhibitor studies are particularly valuable when studying E3 ligases like FBXL17. Treatment with proteasome inhibitors (MG132, bortezomib) or NAE inhibitors (MLN4924) blocks substrate degradation, allowing accumulation and detection of ubiquitinated intermediates. This approach was successfully used to detect endogenous FBXL17-Sufu interaction by stabilizing the complex that would otherwise be transient due to rapid substrate degradation .

Compartment-specific degradation analysis can reveal spatial regulation of FBXL17 activity. This involves subcellular fractionation to separate nuclear and cytoplasmic proteins, followed by degradation assays on specific fractions. Such analysis revealed that SCF FBXL17 degrades SPAST-M1 specifically in the nuclear fraction, highlighting the importance of compartmentalized protein regulation .

For more sophisticated analysis, reconstituted in vitro ubiquitination assays using purified components (E1, E2, SCF-FBXL17 complex, substrate, ubiquitin, ATP) allow direct measurement of ubiquitination activity over time. Western blotting can reveal the formation of poly-ubiquitin chains, while mass spectrometry can identify specific ubiquitination sites on target proteins.

What are the implications of FBXL17 in cancer research, particularly in medulloblastoma models?

FBXL17 has emerged as a significant factor in cancer biology, with particularly strong evidence for its role in medulloblastoma pathogenesis. Its primary oncogenic mechanism involves regulation of the Hedgehog (Hh) signaling pathway through targeted degradation of Suppressor of Fused (Sufu), a critical tumor suppressor. By controlling Sufu degradation, FBXL17 enables Gli1 release and subsequent activation of Hedgehog target genes. Since aberrant Hh pathway activation is a hallmark of certain medulloblastoma subtypes, the "alteration in Fbxl17–Sufu axis" has been identified as "an etiological mechanism of medulloblastoma" .

Experimental evidence from cell models strongly supports this oncogenic role. Studies in DAOY medulloblastoma cells demonstrate that FBXL17 depletion significantly impairs cellular proliferation. This proliferation defect can be rescued either by re-expressing FBXL17 or by simultaneously knocking down Sufu, confirming that FBXL17 promotes medulloblastoma cell proliferation specifically through Sufu degradation .

The in vivo relevance of FBXL17 has been established using orthotopic medulloblastoma models. RNAi-mediated knockdown of FBXL17 in an orthotopic rat model resulted in impaired Hedgehog signaling (evidenced by Sufu accumulation and decreased Gli1 mRNA), marked reduction in tumor progression as monitored by T2-weighted MRI, significantly reduced tumor growth (6.1 ± 1.7 mm² versus 12.4 ± 1.3 mm² in controls), and a substantially lower proliferative index within tumor tissue (5.4 ± 3.6% versus 20.8 ± 2.8% in controls) . These findings provide compelling pre-clinical evidence for FBXL17 as a potential therapeutic target.

Clinical relevance has been demonstrated through the identification of somatic mutations in medulloblastoma patients. Specifically, a somatic mutation in Sufu occurring in medulloblastoma patients with Gorlin syndrome increases Sufu turnover through FBXL17-mediated polyubiquitination, leading to sustained Hedgehog signaling and enhanced cell proliferation . This discovery provides a direct mechanistic link between clinical mutations and aberrant FBXL17-Sufu regulation.

The therapeutic implications of these findings are significant. FBXL17 represents a potential therapeutic target for medulloblastoma and other Hedgehog-driven cancers. Strategies to block FBXL17-mediated degradation of Sufu could inhibit Hedgehog signaling and tumor growth. F-box proteins are increasingly recognized as pharmacological targets in cancer treatment, and development of FBXL17-specific inhibitors could represent a novel therapeutic approach .

What controls should be included when using FBXL17 antibodies for co-immunoprecipitation experiments?

Co-immunoprecipitation (co-IP) experiments with FBXL17 antibodies require rigorous controls to ensure data reliability and interpretability. Input controls are essential, where researchers should reserve a small portion (5-10%) of the pre-cleared lysate before immunoprecipitation. This confirms the presence of target proteins in the starting material and allows quantification of IP efficiency by comparing to precipitated material. This control is critical for interpreting negative results, distinguishing between lack of interaction and technical failure .

Isotype controls represent another crucial element, wherein researchers perform parallel IPs with an antibody of the same isotype but irrelevant specificity. For rabbit polyclonal FBXL17 antibodies, this would typically involve using normal rabbit IgG at an equivalent concentration. This controls for non-specific binding to antibody constant regions and was implicit in studies validating the FBXL17-Sufu interaction .

Beads-only controls should also be implemented by incubating lysate with the precipitation matrix (beads) without antibody. This controls for proteins binding non-specifically to the matrix itself, which is particularly important when using protein A/G beads that may bind endogenous immunoglobulins.

Reciprocal IP represents a powerful validation approach. When studying FBXL17 interaction with specific proteins (such as Sufu or SPAST-M1), researchers should perform reverse IP using antibodies against the interacting partner, which should co-precipitate FBXL17 if the interaction is specific. This approach was successfully used to validate the FBXL17-Sufu interaction in previous studies .

For interactions involving proteins targeted for degradation, proteasome inhibitor treatment is essential. Since FBXL17 targets proteins for degradation, interactions may be transient and difficult to detect. Including samples treated with proteasome inhibitors (e.g., MG132) or NAE inhibitors (MLN4924) stabilizes these interactions by preventing substrate degradation. This approach was crucial for detecting endogenous FBXL17-Sufu interaction in published research .

FBXL17 knockdown/knockout controls provide additional verification. Performing IP in cells where FBXL17 has been depleted by siRNA/shRNA should result in reduced or absent signal, validating antibody specificity and controlling for off-target binding .

How do researchers distinguish between the various protein isoforms of FBXL17 using antibodies?

Differentiating between FBXL17 protein isoforms requires strategic antibody selection and complementary experimental approaches. Epitope-specific antibodies represent the primary strategy, where researchers select or design antibodies targeting regions unique to specific FBXL17 isoforms. Given that FBXL17 has several aliases (FBL17, FBX13, FBXO13) that may represent different isoforms, antibodies targeting the N-terminal region may distinguish full-length isoforms from truncated variants. For maximum specificity, custom antibodies can be generated against isoform-specific junction sequences where exons are alternatively spliced .

Western blotting optimization techniques significantly enhance isoform differentiation. Using high-resolution gels (6-8% polyacrylamide or gradient gels) with extended running times improves separation of closely sized isoforms. Researchers should compare observed molecular weights with the predicted weights of known isoforms and include positive controls of recombinant isoforms expressed in cell lines to aid in identification .

For definitive isoform characterization, immunoprecipitation coupled with mass spectrometry provides unparalleled resolution. This approach involves immunoprecipitating FBXL17 using pan-isoform antibodies, then analyzing the precipitated proteins by mass spectrometry to identify isoform-specific peptides that map to unique regions. This technique can both identify and quantify the relative abundance of different isoforms and has been successfully used to characterize FBXL17 interacting proteins .

Isoform-specific siRNA/shRNA knockdown experiments offer functional validation. By designing silencing RNAs targeting unique regions of specific isoforms and analyzing the differential loss of bands in Western blot, researchers can validate the identity of specific bands corresponding to particular isoforms. This approach has been successfully employed to validate FBXL17 antibody specificity in functional studies .

Subcellular fractionation provides additional discrimination capability, as different isoforms may localize to different cellular compartments. Nuclear/cytoplasmic fractionation can help separate isoforms for individual analysis, as demonstrated in studies revealing compartment-specific functions of FBXL17 in SPAST-M1 regulation .

What technical considerations are important when using FBXL17 antibodies in immunohistochemistry?

When employing FBXL17 antibodies for immunohistochemistry (IHC), several technical considerations are critical for obtaining specific, reproducible results. Antigen retrieval optimization is paramount, as FBXL17 epitopes may be masked during fixation processes. Heat-induced epitope retrieval using citrate buffer (pH 6.0) or EDTA buffer (pH 9.0) should be systematically compared to determine optimal conditions. The type and duration of fixation can significantly impact antibody performance, with over-fixation potentially leading to false-negative results .

Antibody validation in appropriate control tissues represents an essential preliminary step. Researchers should include positive control tissues known to express FBXL17 and negative controls where primary antibody is omitted. Additionally, using tissues from FBXL17 knockdown models or comparing staining patterns with multiple antibodies targeting different FBXL17 epitopes helps confirm staining specificity. The Human Protein Atlas employs such enhanced validation approaches for their antibody characterization .

Optimization of antibody concentration is critical for balancing sensitivity and specificity. Titration experiments with serial dilutions of the primary antibody should be performed to identify the concentration that provides optimal signal-to-background ratio. Commercial FBXL17 antibodies typically work in the 1:100 to 1:500 dilution range for IHC applications, but this must be empirically determined for each experimental system .

Signal detection system selection influences sensitivity and quantification ability. While chromogenic detection (DAB) provides permanent results suitable for archival samples, fluorescent detection offers superior multiplexing capabilities and potentially higher sensitivity for detecting low-level expression. For co-localization studies with potential FBXL17 substrates like Sufu or SPAST-M1, fluorescent multiplexing is particularly advantageous .

Counterstaining and background reduction strategies enhance result interpretability. Nuclear counterstains (hematoxylin for chromogenic detection or DAPI for fluorescence) provide context for evaluating FBXL17 localization, which may be compartment-specific as demonstrated in studies of nuclear SPAST-M1 regulation. To minimize non-specific binding, inclusion of appropriate blocking steps (using serum from the secondary antibody host species) and careful optimization of washing steps are essential .

How can FBXL17 antibodies be used to investigate the role of FBXL17 in neurological disorders?

FBXL17 antibodies provide valuable tools for investigating the emerging role of FBXL17 in neurological disorders, particularly hereditary spastic paraplegia (HSP). For tissue expression profiling, antibodies can map FBXL17 distribution in normal versus pathological neural tissues using immunohistochemistry or immunofluorescence. This approach helps identify specific neuronal populations and brain regions where FBXL17 dysregulation may contribute to disease pathology. The compartment-specific effects of FBXL17, particularly its nuclear activity in degrading SPAST-M1, make subcellular localization studies especially informative .

Protein interaction studies represent a critical application area. Co-immunoprecipitation with FBXL17 antibodies followed by immunoblotting for Spastin (SPAST) can reveal their physical association in neural cells. Proximity ligation assays provide in situ visualization of this interaction with subcellular resolution. These approaches have successfully demonstrated that FBXL17 interacts with SPAST-M1 specifically via the BTB domain at its N-terminus, and that disease-associated mutations like SPAST Y52C disrupt this interaction .

For understanding the disease mechanisms, analyzing protein degradation dynamics is essential. FBXL17 antibodies can track SPAST-M1 protein levels in response to FBXL17 manipulation in neuronal cells. Cycloheximide chase assays using these antibodies reveal altered SPAST-M1 half-life in disease models or when FBXL17 is inhibited. Research has demonstrated that inhibition of SCF FBXL17 E3 ubiquitin ligase activity decreases proteasome-dependent degradation of SPAST-M1 and induces axonal extension, potentially counteracting HSP pathology .

In patient-derived samples, FBXL17 antibodies can assess potential dysregulation in affected individuals. Comparing FBXL17 and SPAST-M1 levels in control versus patient samples (from biopsies, iPSC-derived neurons, or post-mortem tissue) may reveal aberrant protein regulation associated with disease status. The inverse correlation between FBXL17 and SPAST-M1 protein levels observed in vitro and during embryonic development suggests this relationship may be altered in pathological conditions .

For therapeutic development applications, FBXL17 antibodies can evaluate the efficacy of potential treatments targeting the FBXL17-SPAST axis. Monitoring changes in SPAST-M1 levels and localization following treatment with small molecule inhibitors of SCF FBXL17 E3 ubiquitin ligase provides quantitative measures of target engagement. Studies have shown that inhibiting FBXL17 can rescue phenotypic defects caused by SPAST mutations, suggesting a promising therapeutic approach .

What is the role of FBXL17 in the quality control of BTB domain-containing proteins?

FBXL17 serves as a critical component in the quality control mechanisms ensuring proper dimerization of BTB (Broad-Complex, Tramtrack and Bric-a-brac) domain-containing proteins. As a substrate-recognition component of the SCF(FBXL17) E3 ubiquitin ligase complex, FBXL17 specifically participates in a specialized quality control pathway known as dimerization quality control (DQC). This process is essential for maintaining the functional integrity of the numerous BTB domain-containing proteins that regulate diverse cellular processes .

The molecular recognition mechanism involves FBXL17's ability to specifically recognize and bind to a conserved degron of non-consecutive residues. Crucially, this degron is present at the interface of BTB dimers that have aberrant composition, essentially allowing FBXL17 to distinguish between properly formed functional dimers and defective ones. Once bound to these aberrant BTB dimers, the SCF(FBXL17) complex facilitates their ubiquitination, targeting them for degradation by the proteasome .

This quality control function has significant physiological implications, particularly in neural development and function. Research indicates that the ability of the SCF(FBXL17) complex to eliminate compromised BTB dimers is required for the differentiation and survival of neural crest and neuronal cells. This suggests that proper BTB protein dimerization, monitored and maintained by FBXL17, is essential for neuronal health .

The importance of this quality control mechanism is further highlighted by the FBXL17-SPAST interaction in neurological disease. SPAST-M1 (Spastin) contains a BTB domain at its N-terminus through which it specifically interacts with FBXL17. A disease-causing mutation, SPAST Y52C, located in this BTB domain, prevents interaction with FBXL17, thereby escaping this quality control mechanism. This results in aberrant protein quantity and loss of functionality, contributing to hereditary spastic paraplegia pathology .

Beyond neurological implications, the BTB quality control function of FBXL17 likely extends to other cellular contexts where BTB domain-containing proteins play regulatory roles, including transcription regulation, cytoskeletal organization, and ion channel function. This suggests that FBXL17 dysfunction could have wide-ranging impacts on cellular homeostasis through disruption of this critical quality control mechanism.