FBXW5 Antibody

What is FBXW5 and what cellular functions does it regulate?

FBXW5 (F-box and WD repeat domain containing 5) is a critical component of ubiquitin-mediated protein degradation pathways. It belongs to the F-box protein family, characterized by an approximately 40 amino acid motif that serves as a recognition component in SCF (Skp1, Cullin, F-box) ubiquitin ligase complexes . FBXW5 contains WD40 repeats that are essential for substrate recognition and binding, particularly for interactions with proteins like LATS1 . Functionally, FBXW5 plays pivotal roles in targeting specific proteins for ubiquitination and subsequent proteasomal degradation, thereby regulating various cellular processes including cell cycle progression, apoptosis, and signaling pathways such as the Hippo pathway .

What are the common applications for FBXW5 antibodies in research?

FBXW5 antibodies are utilized across multiple experimental approaches. Western blotting (WB) represents a primary application, with recommended dilutions ranging from 1:500 to 1:3000 depending on sample type and antibody sensitivity . Immunohistochemistry (IHC) is another major application, with suggested dilutions between 1:50 and 1:500 . Additionally, FBXW5 antibodies have been employed in immunoprecipitation experiments to investigate protein-protein interactions, particularly with binding partners such as LATS1 in the Hippo signaling pathway . ELISA techniques are also mentioned in the applications spectrum . These methodologies collectively enable researchers to detect, quantify, and localize FBXW5 in various experimental and clinical samples, facilitating investigations into its role in normal cellular processes and disease mechanisms.

What tissue and cell types show notable FBXW5 expression?

FBXW5 expression has been documented across multiple tissue and cell types. In cell culture models, FBXW5 is expressed in human embryonic kidney cells (HEK-293) and the K-562 leukemia cell line, making these suitable positive controls for antibody validation . Multiple gastric cancer cell lines including MKN1, CLS145, AGS, SNU1, HGC-27, MGC-803, and BGC-823 demonstrate varying levels of FBXW5 expression, with generally higher expression in cancer cells compared to normal gastric epithelial cells like GES-1 . In tissue samples, FBXW5 antibodies have successfully detected the protein in mouse testis and human kidney tissues using immunohistochemistry . In gastric cancer clinical specimens, FBXW5 is predominantly localized to the cytoplasm, and elevated expression correlates with advanced tumor characteristics and poorer patient prognosis .

What are the recommended storage and handling conditions for FBXW5 antibodies?

FBXW5 antibodies should be stored at -20°C, where they remain stable for approximately one year after shipment . The standard storage buffer composition consists of PBS with 0.02% sodium azide and 50% glycerol at pH 7.3 . For small volume formats (20μl), the preparations typically contain 0.1% BSA as a stabilizer . Unlike some antibody preparations, aliquoting is generally unnecessary for -20°C storage of these reagents . When working with these antibodies, it is advisable to minimize freeze-thaw cycles, maintain cold chain during handling, and adhere to the manufacturer's specific recommendations for dilution and application parameters. These storage and handling guidelines help preserve antibody activity and specificity, ensuring consistent experimental results across multiple investigations.

How does FBXW5 regulate the Hippo signaling pathway in cancer progression?

FBXW5 serves as a critical negative regulator of the tumor-suppressive Hippo signaling pathway through a specific molecular mechanism involving LATS1 degradation. Research has demonstrated that FBXW5 directly binds to LATS1, a core kinase in the Hippo pathway, through its WD40 domain . This interaction facilitates the ubiquitination and subsequent proteasomal degradation of LATS1, effectively shortening its half-life . The reduced LATS1 levels lead to decreased phosphorylation of the transcriptional co-activator YAP1 (Yes-associated protein 1), allowing unphosphorylated YAP1 to translocate into the nucleus . Nuclear YAP1 then activates transcription of target genes like CTGF (Connective Tissue Growth Factor), promoting cellular proliferation, invasion, and metastasis . Importantly, silencing FBXW5 in gastric cancer cells increases LATS1 and phosphorylated YAP1 levels while decreasing total YAP1 and CTGF expression, confirming the causal relationship . Clinically, a positive correlation between FBXW5 and YAP1 expression in gastric cancer patient samples further validates this regulatory mechanism .

What methodologies are effective for studying FBXW5-mediated protein ubiquitination?

Investigating FBXW5-mediated protein ubiquitination requires a multi-faceted experimental approach. Co-immunoprecipitation (Co-IP) assays using FBXW5 antibodies represent a foundational technique for identifying interaction partners, as demonstrated in studies showing direct binding between FBXW5 and LATS1 . For domain-specific interactions, deletion mutants of the F-box motif and WD40 domain (ΔF-box and ΔWD) can be employed in Co-IP experiments to define the precise binding interfaces; such approaches revealed that the WD40 repeats of FBXW5 are essential for LATS1 interaction . To assess protein stability and turnover rates, cycloheximide (CHX) chase assays combined with western blotting enable researchers to track degradation kinetics of target proteins in the presence or absence of FBXW5 . For direct examination of ubiquitination, in vivo ubiquitination assays involving co-transfection of tagged ubiquitin, target protein, and FBXW5 constructs, followed by immunoprecipitation under denaturing conditions and western blotting, provide definitive evidence of FBXW5-dependent ubiquitination . Additionally, proteasome inhibitors like MG132 can confirm the involvement of proteasomal degradation in the observed protein turnover .

What is the relationship between FBXW5 and chemoresistance in cancer cells?

FBXW5 significantly contributes to chemoresistance in cancer cells through multiple mechanisms linked to its effects on cell survival pathways. In gastric cancer models, FBXW5 expression levels directly correlate with resistance to standard chemotherapeutic agents. Cell viability assays using gradient concentrations of 5-fluorouracil (0-160 μM) demonstrated that downregulation of FBXW5 markedly increased cancer cell sensitivity to the drug, while FBXW5 upregulation had the opposite effect, enhancing resistance . Similar patterns were observed with cisplatin treatment . Clonogenic assays further validated these findings, showing that FBXW5 silencing in MGC-803 cells led to a 62% reduction in colony formation following 5-fluorouracil treatment, compared to only a 42% reduction in control cells . The molecular basis for this chemoresistance appears linked to FBXW5's inhibitory effect on the Hippo pathway through LATS1 degradation, which promotes YAP1 nuclear translocation and activation of anti-apoptotic programs . Additionally, FBXW5's influence on cell survival is reflected in its regulation of apoptotic markers, where its knockdown increases caspase-3 activity, downregulates the anti-apoptotic protein survivin, and upregulates the cell cycle inhibitor p21 .

What are the optimal protocols for FBXW5 detection by western blotting?

Western blotting for FBXW5 requires careful optimization to ensure specific and sensitive detection. Based on the antibody specifications, recommended dilution ranges for FBXW5 detection fall between 1:500 and 1:3000, though this should be titrated for each experimental system and sample type . The expected molecular weight for FBXW5 is approximately 64-65 kDa, which serves as a critical validation parameter . For positive controls, HEK-293 or K-562 cells have shown reliable FBXW5 expression and can be incorporated as reference standards . When preparing samples, standard RIPA buffer extraction is generally effective, though phosphatase inhibitors should be included when examining phosphorylation-dependent interactions. For optimal protein separation, 8-10% SDS-PAGE gels are recommended given the target's molecular weight. During transfer, PVDF membranes may offer advantages over nitrocellulose for proteins in this size range. For blocking, either 5% non-fat milk or BSA in TBST can be used, with the latter preferred when probing for phosphorylated proteins in the same pathway. Primary antibody incubation should proceed overnight at 4°C for maximum sensitivity and specificity. After thorough washing, HRP-conjugated anti-rabbit secondary antibodies at 1:5000-1:10000 dilutions are appropriate for detection systems.

How should researchers design siRNA experiments to study FBXW5 function?

Designing effective siRNA experiments for FBXW5 functional studies requires careful consideration of multiple factors. First, researchers should design or select at least 2-3 different siRNA sequences targeting distinct regions of FBXW5 mRNA to mitigate off-target effects and confirm phenotypic consistency . Based on successful previous studies, transfection of 20-50 nM siRNA using standard lipid-based transfection reagents in gastric cancer cell lines like MKN1 has yielded effective knockdown . Validation of knockdown efficiency is critical and should be performed at both mRNA level (via qRT-PCR) and protein level (via western blotting) 48-72 hours post-transfection . Non-targeting siRNA sequences with similar GC content must be used as controls to account for non-specific effects of the transfection process . For functional assays, cell proliferation can be monitored using methods like CCK-8 assays, while apoptosis should be assessed using complementary approaches such as Annexin V staining and caspase-3 activity assays to provide comprehensive data . Cell cycle analysis via flow cytometry following PI staining helps determine specific phase alterations induced by FBXW5 knockdown . For migration studies, both wound healing and transwell assays offer valuable complementary data .

What are the critical parameters for successful immunohistochemical detection of FBXW5 in tissue samples?

Successful immunohistochemical detection of FBXW5 in tissue samples depends on several critical parameters that must be carefully optimized. Antigen retrieval represents a key step, with recommended protocols including Tris-EDTA (TE) buffer at pH 9.0, though citrate buffer at pH 6.0 provides an alternative option . The optimal antibody dilution range spans from 1:50 to 1:500, necessitating titration for each specific tissue type and fixation condition . Positive control tissues validated for FBXW5 detection include mouse testis and human kidney samples, which should be incorporated into experimental design to verify staining efficacy . For gastric cancer tissue analysis, researchers should note that FBXW5 demonstrates predominantly cytoplasmic localization, providing an important reference for evaluating staining patterns . Detection systems employing biotin-streptavidin amplification have proven effective in previous studies, though polymer-based detection methods may offer advantages in terms of reduced background. Semi-quantitative scoring systems incorporating both staining intensity and percentage of positive cells can be employed for comparative analyses, with thresholds for "high" versus "low" expression established based on median values or statistical optimization relative to clinical parameters .

How can researchers effectively investigate FBXW5-mediated regulation of actin dynamics?

Investigation of FBXW5's role in actin dynamics requires a comprehensive experimental approach combining morphological, biochemical, and functional analyses. Immunofluorescence staining of F-actin using phalloidin represents a foundational technique, enabling visualization of actin stress fiber organization, polymerization states, and cytoskeletal rearrangements in response to FBXW5 manipulation . Quantitative biochemical assessment through fractionation of F-actin (filamentous) and G-actin (globular) followed by western blotting allows researchers to determine the F-actin to total actin ratio, providing objective measurement of polymerization status . For mechanistic investigations, western blot analysis of RhoA-ROCK1-pMLC2 signaling components following FBXW5 knockdown or overexpression reveals key regulatory pathways, as previous studies demonstrated significant reduction in RhoA, ROCK1, and phosphorylated MLC2 expression upon FBXW5 depletion . Activity assays for small GTPases including Cdc42 and Rac1 should be incorporated to comprehensively assess the impact on cytoskeletal regulatory pathways . Functional consequences can be evaluated through migration assays such as wound healing or transwell chambers, with FBXW5 knockdown previously shown to reduce migratory potential while overexpression enhanced this capacity .

How should researchers interpret contradictory FBXW5 expression data across different cancer types?

When encountering contradictory FBXW5 expression patterns across cancer types, researchers should implement a structured analytical approach. First, evaluate methodological differences in detection techniques (antibody clones, detection platforms, scoring systems) that might contribute to discrepancies. Tissue-specific contexts must be considered, as FBXW5 may have divergent roles depending on the cellular environment and predominant signaling networks in different tissues. Previous research has established FBXW5 overexpression in gastric cancer correlating with poor outcomes, but this pattern may not universally apply to all malignancies . Researchers should examine the status of key FBXW5 substrates across cancer types, particularly LATS1 and components of the Hippo pathway, as the functional significance of FBXW5 expression may depend on substrate availability and activation states . Analysis should incorporate genetic and epigenetic alterations affecting the FBXW5 locus (chromosome 9q33.2) across cancer types, as these might explain expression differences. Additionally, contextual evaluation of F-box protein family member expression is valuable, as compensatory mechanisms involving related F-box proteins may influence the net effect of FBXW5 alterations. When possible, multi-omics integration combining transcriptomic, proteomic, and phosphoproteomic data provides the most comprehensive understanding of FBXW5's role across cancer types.

How can researchers differentiate between direct and indirect effects of FBXW5 on cellular phenotypes?

Distinguishing between direct and indirect effects of FBXW5 on cellular phenotypes requires a systematic experimental strategy. Rescue experiments represent a critical approach, where phenotypes induced by FBXW5 knockdown or overexpression are assessed following restoration or depletion of putative downstream targets. Previous research employed this strategy by blocking LATS1-YAP1 in FBXW5-manipulated cells, demonstrating that FBXW5-mediated regulation of the Hippo pathway was partially responsible for observed phenotypes . Temporal analyses tracking protein expression, post-translational modifications, and phenotypic changes at multiple time points after FBXW5 manipulation help establish causative sequences and identify primary versus secondary effects. Domain-specific mutations offer targeted insights, as demonstrated by experiments with deletion mutants of the F-box motif and WD40 domain (ΔF-box and ΔWD) that identified the WD40 repeats as essential for LATS1 interaction . Proximity labeling techniques such as BioID or APEX can identify the immediate protein neighborhood of FBXW5, distinguishing direct binding partners from downstream effectors. For definitive substrate identification, in vitro ubiquitination assays using purified components (E1, E2, Cullin, Skp1, FBXW5, and candidate substrates) can demonstrate direct enzymatic activity. Integration with global approaches such as proteomics following FBXW5 manipulation, particularly with proteasome inhibition, can identify the broader spectrum of proteins affected either directly or indirectly.

What controls and validation steps are essential when evaluating FBXW5 antibody specificity?

Rigorous validation of FBXW5 antibody specificity requires multiple complementary approaches. Positive and negative control samples are fundamental, with HEK-293 and K-562 cells serving as validated positive controls for western blotting applications . For negative controls, FBXW5 knockout or knockdown samples generated via CRISPR-Cas9 or siRNA technologies provide the most stringent verification . Cross-reactivity assessment against related F-box family proteins is essential, particularly those sharing structural similarities in the F-box or WD40 domains. Multiple detection methods should be employed to confirm specificity across different applications, including western blotting, immunohistochemistry, and immunofluorescence, as antibodies may perform differently depending on protein conformation and sample preparation . Peptide competition assays, where pre-incubation of the antibody with the immunogen peptide should abolish specific signals, provide additional validation of binding specificity. Molecular weight verification is critical, with FBXW5 expected at approximately 64-65 kDa . When possible, detection with multiple antibodies targeting different epitopes of FBXW5 offers powerful cross-validation. For clinical applications, correlation between protein detection methods (e.g., IHC) and mRNA expression data from the same samples provides important confirmation of specificity in complex tissue environments .

What are common challenges in FBXW5 protein detection and how can they be overcome?

Researchers frequently encounter several challenges when detecting FBXW5 protein that require specific optimization strategies. Background signal issues in western blotting can be addressed by increasing blocking stringency (5% BSA instead of milk), extending blocking duration to 2 hours, and using freshly prepared TBST with 0.1% Tween-20 for more effective washing . For weak signal problems, researchers should first verify sample integrity using housekeeping controls, then consider enhanced chemiluminescence substrates, extended primary antibody incubation at 4°C overnight, and concentration of protein lysates to increase target abundance . Multiple band detection commonly occurs with F-box proteins due to post-translational modifications or degradation intermediates; researchers should compare observed patterns with positive control samples (HEK-293, K-562) and consider phosphatase treatment to determine if phosphorylation contributes to band shifts . For immunohistochemistry applications, inconsistent staining often responds to optimized antigen retrieval using TE buffer at pH 9.0 as recommended, though citrate buffer at pH 6.0 provides an alternative . When initial detection attempts fail, researchers should verify antibody viability through dot blot analysis of the immunizing peptide and consider testing the antibody on overexpression systems before proceeding to endogenous detection.

How should researchers troubleshoot unexpected results in FBXW5 functional studies?

When encountering unexpected results in FBXW5 functional studies, researchers should implement a systematic troubleshooting approach. First, thoroughly validate knockdown or overexpression efficiency at both protein and mRNA levels using multiple detection methods, as incomplete manipulation may yield inconsistent phenotypes . Cell line heterogeneity can significantly impact outcomes; researchers should verify results across multiple cell lines and consider single-cell cloning if heterogeneous responses are observed . For contradictory phenotypes between different experimental systems, analyze the baseline expression levels of key FBXW5 substrates and pathway components (particularly LATS1, YAP1, and other Hippo pathway members), as contextual differences may explain divergent functional outcomes . Careful timing analysis is essential, as early and late effects of FBXW5 manipulation may differ substantially; time-course experiments capturing multiple endpoints can resolve apparent contradictions . Off-target effects represent a common concern with siRNA approaches; researchers should compare phenotypes across multiple independent siRNA sequences targeting FBXW5 and consider rescue experiments with siRNA-resistant constructs . When manipulations affect cellular viability, carefully distinguish between direct effects on proliferation, indirect consequences of apoptosis, and potential cell cycle perturbations using complementary assays such as BrdU incorporation, Annexin V staining, and cell cycle analysis .

What strategies can improve reproducibility in co-immunoprecipitation experiments with FBXW5?

Enhancing reproducibility in FBXW5 co-immunoprecipitation experiments requires attention to several critical factors. Lysis buffer optimization is paramount; for studying FBXW5-substrate interactions, use buffers containing 1% NP-40 or Triton X-100 with moderate salt concentrations (150mM NaCl) to preserve protein-protein interactions while minimizing non-specific binding . Proteasome inhibitors (e.g., MG132, 10μM for 4-6 hours before lysis) should be routinely included to stabilize transient interactions between FBXW5 and its ubiquitination targets . For FBXW5's interaction with SCF complex components, consider crosslinking approaches to capture these associations more effectively. Antibody selection significantly impacts results; when studying endogenous interactions, use antibodies validated specifically for immunoprecipitation applications, potentially different from those optimized for western blotting . For tagged-protein approaches, epitope tags (FLAG, Myc) placed at the N-terminus of FBXW5 minimize interference with C-terminal substrate recognition via WD40 repeats . Pre-clearing lysates with protein A/G beads reduces non-specific binding, while extensive washing (at least 4-5 washes) with buffers of increasing stringency improves signal-to-noise ratio. Negative controls must include IgG matched to the host species of the immunoprecipitating antibody, while positive controls should verify the presence of known FBXW5 interactors such as SKP1 . Reciprocal co-immunoprecipitation, pulling down from both directions, provides the strongest evidence for specific interactions.

How can researchers optimize FBXW5 overexpression systems for functional studies?

Optimizing FBXW5 overexpression systems for functional studies requires careful consideration of multiple technical aspects. Expression vector selection is crucial, with consideration for promoter strength; while CMV promoters provide high expression levels suitable for biochemical studies, weaker promoters like EF1α may better approximate physiological expression for functional analyses . For stable expression systems, lentiviral vectors have demonstrated effectiveness in gastric cancer cell models, offering consistent expression and the ability to select stable populations . Epitope tag placement significantly impacts FBXW5 function; N-terminal tags (Myc-DDK as used in previous studies) generally preserve the substrate-binding capacity of the C-terminal WD40 domain . Expression level verification is essential through both western blotting and qRT-PCR, comparing to endogenous levels in appropriate positive control cells . When studying FBXW5's E3 ligase functions, co-expression of core SCF components may be necessary in some cell systems to reconstitute full activity. For phenotypic studies, inducible expression systems using tetracycline-responsive elements provide valuable tools to distinguish between acute and chronic effects of FBXW5 overexpression. Functional mutants serve as critical controls, particularly those lacking the F-box domain (disrupting SCF complex integration) or with mutations in the WD40 repeats (impairing substrate recognition) . Finally, careful selection of appropriate control vectors (empty vector with identical backbone) is essential for accurate interpretation of overexpression phenotypes.

How might FBXW5 serve as a therapeutic target in cancer treatment?

FBXW5 represents a promising therapeutic target in cancer treatment based on its central role in promoting tumor progression, metastasis, and chemoresistance. Several strategic approaches could exploit FBXW5's vulnerabilities. Small molecule inhibitors designed to disrupt the FBXW5-LATS1 interaction represent a direct targeting strategy, focusing on the critical WD40 repeats that mediate substrate binding . Proteolysis-targeting chimeras (PROTACs) offer an alternative approach, potentially redirecting FBXW5's ubiquitin ligase activity toward self-destruction. Synthetic peptides mimicking the binding interface between FBXW5 and SCF complex components could disrupt E3 ligase assembly, inhibiting FBXW5's catalytic function. Importantly, therapeutic strategies targeting FBXW5 would directly impact the Hippo signaling pathway, potentially reactivating this tumor-suppressive cascade by stabilizing LATS1, increasing YAP1 phosphorylation, and preventing nuclear translocation of this oncogenic transcriptional co-activator . Combination therapies pairing FBXW5 inhibition with standard chemotherapeutics appear particularly promising, as FBXW5 knockdown significantly enhances cancer cell sensitivity to both 5-fluorouracil and cisplatin . Pre-clinical models support FBXW5's therapeutic relevance, as demonstrated by the significant impairment of xenograft tumor growth following FBXW5 silencing, with corresponding decreases in markers of proliferation (Ki-67), angiogenesis (CD31), and epithelial-mesenchymal transition (N-cadherin) .

What role might FBXW5 play in cancer cell response to targeted therapies?

FBXW5's potential influence on cancer cell response to targeted therapies extends beyond conventional chemotherapeutics to include pathway-specific inhibitors and emerging therapeutic modalities. Given FBXW5's established role in modulating the Hippo signaling pathway through LATS1 degradation, it likely impacts response to YAP inhibitors currently in development . High FBXW5 expression could confer resistance to these agents by maintaining YAP1 nuclear localization despite inhibitor presence . FBXW5's involvement in regulating actin dynamics via the RhoA-ROCK1-pMLC2 signaling axis suggests potential influence on response to cytoskeletal-targeting agents, including ROCK inhibitors . The protein's contribution to apoptotic resistance, evidenced by its knockdown increasing caspase-3 activity and reducing survivin expression, indicates it may modulate response to BH3 mimetics and other apoptosis-inducing targeted agents . As a component of SCF ubiquitin ligase complexes, FBXW5 may also influence response to proteasome inhibitors like bortezomib, potentially through accumulation of its substrates when proteasomal degradation is blocked . Investigation of these interactions would benefit from combination therapy studies in preclinical models, correlation of FBXW5 expression with clinical response data, and mechanistic studies defining how FBXW5-mediated pathways intersect with the molecular targets of specific therapeutic agents.

How can advanced imaging techniques enhance our understanding of FBXW5 dynamics in live cells?

Advanced imaging technologies offer unprecedented opportunities to elucidate FBXW5's dynamic behavior in living cells. Fluorescence resonance energy transfer (FRET) approaches utilizing fluorescently tagged FBXW5 and substrate proteins can visualize real-time interactions in living cells, particularly valuable for capturing the transient nature of E3 ligase-substrate binding . Photo-activatable or photo-switchable fluorescent protein fusions with FBXW5 enable pulse-chase imaging to track protein movement and turnover rates within specific cellular compartments. Super-resolution microscopy techniques such as structured illumination microscopy (SIM) or stochastic optical reconstruction microscopy (STORM) can reveal FBXW5's precise subcellular localization relative to substrates, SCF complex components, and proteasomes at nanometer resolution. For functional studies, optogenetic approaches permit spatiotemporally controlled activation or inhibition of FBXW5, allowing researchers to dissect local versus global effects of its activity. Live-cell ubiquitination sensors paired with FBXW5 visualization could demonstrate the kinetics of substrate ubiquitination in real time. These advanced imaging approaches would complement traditional biochemical analyses by providing spatial and temporal dimensions to our understanding of FBXW5 biology, potentially revealing microenvironment-dependent functions and rapid dynamic changes that static methods cannot capture.

What computational approaches can identify novel FBXW5 substrates and interaction partners?

Computational methods offer powerful avenues for discovering novel FBXW5 substrates and interaction partners. Structure-based prediction approaches beginning with crystal structure analysis of FBXW5's WD40 domain can identify substrate recognition motifs and enable in silico screening for proteins containing these sequences . Machine learning algorithms trained on known F-box protein-substrate interactions can predict novel FBXW5 targets based on sequence features, structural characteristics, and expression correlation patterns. Integrative network analysis incorporating protein-protein interaction databases, co-expression networks, and pathway enrichment can identify functional clusters associated with FBXW5, highlighting potential new interactors. Degradome analysis comparing proteome changes following FBXW5 manipulation with corresponding transcriptome data can distinguish proteins whose abundance is post-transcriptionally regulated, a signature of potential ubiquitination targets . Evolutionary conservation analysis focusing on predicted substrate binding regions across species can identify functionally important interaction interfaces. For validation of computationally predicted interactions, researchers should employ scaffolding approaches that integrate predictions from multiple algorithms with different theoretical foundations, prioritizing candidates with support from diverse methods. Experimentally, these computational predictions should be verified through focused biochemical approaches including co-immunoprecipitation, in vitro binding assays, and ubiquitination studies .