WBP2 Human

What is WBP2 and what is its molecular structure?

WBP2 (WW Domain Binding Protein 2) is a protein coding gene that functions as a transcriptional coactivator. Structurally, WBP2 contains PPxY (PY) motifs at its C-terminal domain that facilitate binding to proteins containing WW domains. The protein interactions of WBP2 occur primarily through these PY motifs, with different motifs showing specificity for different binding partners. For example, the PY2 and PY3 motifs are vital for WW domain binding interactions with TAZ and YAP, respectively, while the binding of WWC3 is linked to all three PY motifs of WBP2 .

Recent evidence indicates that WBP2 can also interact with proteins lacking the WW domain, such as LATS2 and Pax8, suggesting that WBP2's protein interactions extend beyond the classical PY-WW interactions . Additionally, the binding of neural precursor cells to the PR domain of WBP2 suggests alternative modes of transcriptional regulation .

What is the historical background of WBP2 research?

WBP2 was initially identified as a partner of Yes-associated Protein (YAP) in 1995. Despite being discovered over two decades ago, significant momentum in WBP2 research has only accelerated in recent years . The first ten years following its discovery primarily focused on biochemistry aspects, while the subsequent decade saw increasing emphasis on WBP2's involvement in signal transduction pathways .

A significant breakthrough came in 2007 when WBP2 was first associated with breast cancer. Phosphoproteomic profiling revealed WBP2 to be hyperphosphorylated in an isogenic MCF-10AT breast cancer progression model . This discovery opened new avenues of research into WBP2's role in cancer biology.

How does WBP2 function in normal cellular processes?

In normal cellular contexts, WBP2 acts as a transcriptional coactivator of estrogen and progesterone receptors (ESR1 and PGR) upon hormone activation. In the presence of estrogen, WBP2 binds to ESR1-responsive promoters and is required for YAP1 coactivation function on PGR activity . It also synergizes with other proteins in enhancing PGR activity and modulates expression of post-synaptic scaffolding proteins via regulation of ESR1, ESR2, and PGR .

Beyond its role in cancer, WBP2 is involved in multiple physiological processes. It can cause hearing impairment when defective and is associated with neurological diseases like Huntington's disease and Alzheimer's disease . In the endocrine system, WBP2 participates in insulin signaling and lipid metabolism, and may be involved in thyroid differentiation in conjunction with Pax8 . It also plays roles in inflammatory responses through T cell regulation and in reproductive system development through oocyte activation .

How does WBP2 contribute to cancer progression?

WBP2 has emerged as a key node connecting multiple signaling pathways associated with cancer, including ER/PR, EGFR, PI3K, Hippo, and Wnt pathways . This interconnection enables WBP2 to influence various hallmarks of cancer.

In breast cancer, WBP2 has been shown to promote oncogenic properties such as anchorage-independent growth and invasiveness in breast epithelial cells . Its expression is higher in cancer tissues compared to normal tissues, and it contributes to malignant development and clinical drug resistance .

In lung cancer, WBP2 is highly expressed in cancer specimens and cell lines, with expression closely related to advanced pTNM stage, lymph node metastasis, and poor prognosis . Experimental evidence demonstrates that WBP2 significantly promotes the proliferation and invasion of lung cancer cells both in vivo and in vitro .

What is the role of WBP2 in the Hippo signaling pathway?

WBP2 plays a critical role in regulating the Hippo pathway, particularly in lung cancer. Research has revealed a unique mechanism through which WBP2 negatively regulates this pathway:

Wild-type WBP2 competitively binds to the WW domain of WWC3 (WW and C2 domain-containing-3) with LATS1 (Large tumor suppressor-1) through its PPxY motifs. This competitive binding inhibits the formation of the WWC3-LATS1 complex, reducing the phosphorylation level of LATS1 and suppressing the activity of the Hippo pathway. This ultimately promotes YAP nuclear translocation .

This mechanism is particularly significant because it shows that WBP2 promotes the malignant phenotype of lung cancer cells in a manner that is not directly dependent upon YAP, providing insights into upstream regulation of Hippo signaling .

What evidence exists for WBP2's role as a potential biomarker in cancer?

Translational research supports WBP2 as a biomarker for early detection, prognosis, and companion diagnostics, particularly in breast cancer . The evidence includes:

• Differential expression: WBP2 is highly expressed in various cancer specimens, including breast and lung cancers, compared to normal tissues .

• Correlation with clinical outcomes: In lung cancer, WBP2 expression is closely related to advanced pTNM stage, lymph node metastasis, and poor prognosis .

• Functional significance: WBP2 promotes malignant phenotypes in multiple cancer types and contributes to drug resistance in breast cancer .

How does WBP2 interact with other oncogenic pathways?

WBP2 functions as an integrator of multiple signaling networks in cancer cells. Key interactions include:

• ER/PR Pathway: WBP2 acts as a transcriptional coactivator of estrogen and progesterone receptors (ESR1 and PGR), enhancing their activity upon hormone activation .

• PI3K/Akt Pathway: WBP2 is implicated in insulin signaling and the PI3K/Akt pathway. There is evidence that USF-1 and WBP2 may act as causative factors in insulin dysregulation-associated cancers because the insulin and PI3K/Akt pathways control USF-1 phosphorylation .

• Hippo Pathway: As detailed earlier, WBP2 negatively regulates the Hippo pathway by inhibiting the formation of the WWC3-LATS1 complex .

• Wnt Pathway: WBP2 has connections to the Wnt pathway, although the mechanisms are less thoroughly characterized in the available search results .

• EGFR Pathway: WBP2 is linked to EGFR signaling, which is particularly relevant in cancers where EGFR overexpression or mutation drives progression .

How is WBP2 regulated at the post-transcriptional level?

WBP2 is subject to significant post-transcriptional regulation, particularly by microRNAs (miRNAs). These small non-coding RNAs bind to the 3' untranslated regions (3' UTRs) of WBP2 mRNA to adjust gene expression through either translational inhibition or mRNA degradation .

Several miRNAs have been identified as regulators of WBP2:

• miR-23a, miR-206, and miR-613 in breast cancer

• miR-485-5p in hepatocellular carcinoma (HCC)

All these miRNAs downregulate WBP2 expression by targeting its 3' UTRs .

Interestingly, there is also a reverse regulatory mechanism where WBP2 can affect miRNA biogenesis. Research by Tabatabaeian et al. demonstrated that WBP2 inhibits the oncogenic properties of DiGeorge Critical Region 8 (DGCR8), an essential component of the microprocessor complex, leading to disruption of downstream mature miRNA formation . The exact mechanism of this process remains unclear, but it may involve physical binding effects or indirect participation in signaling pathways.

What post-translational modifications affect WBP2 function?

WBP2 undergoes several post-translational modifications that regulate its activity and interactions. One significant modification is phosphorylation, which was identified in the initial association of WBP2 with breast cancer. Phosphoproteomic profiling revealed WBP2 to be hyperphosphorylated in breast cancer progression models .

Additionally, WBP2 can be regulated through ubiquitination. It has been shown that WBP2 can be promoted to degradation by itchy E3 ubiquitin-protein ligase (ITCH), which attenuates the proliferation of CD4+ T cells and participates in the inflammatory response . This degradation mechanism represents an important control point for WBP2 protein levels and activity.

Other potential post-translational modifications and their effects on WBP2 function remain areas for further research.

What experimental approaches are most effective for studying WBP2 regulation?

When investigating WBP2 regulation, researchers should consider multiple complementary approaches:

• Gene Expression Analysis:

  • qRT-PCR for mRNA quantification

  • RNA-seq for transcriptome-wide effects

  • In situ hybridization for tissue localization

• Protein Analysis:

  • Western blotting for protein levels and phosphorylation status

  • Co-immunoprecipitation for protein-protein interactions

  • Mass spectrometry for post-translational modifications

  • Immunohistochemistry for tissue expression patterns

• Functional Analysis:

  • Gain- and loss-of-function experiments (overexpression and knockdown/knockout)

  • Luciferase reporter assays for transcriptional activity

  • CHIP-seq for chromatin interactions

• miRNA Regulation:

  • Luciferase reporter assays with wild-type and mutated 3'UTR constructs

  • miRNA mimics and inhibitors

  • AGO-RIP (Argonaute RNA immunoprecipitation) to identify miRNA-mRNA interactions

• Post-translational Modifications:

  • Phospho-specific antibodies

  • Phosphoproteomics

  • Ubiquitination assays

  • Protein stability assays

Recent studies have successfully employed gain- and loss-of-function experiments in both in vitro and in vivo models to assess WBP2's role in cancer progression, particularly in lung cancer research .

What mechanisms underlie WBP2's role in breast cancer progression?

WBP2 contributes to breast cancer progression through multiple mechanisms:

• Transcriptional Coactivation: WBP2 acts as a transcriptional coactivator of estrogen and progesterone receptors (ER/PR), enhancing hormone-induced gene expression that promotes cancer cell proliferation and survival .

• Signal Pathway Integration: WBP2 serves as a node connecting multiple oncogenic pathways, including ER/PR, EGFR, PI3K, Hippo, and Wnt signaling .

• Promotion of Cancer Hallmarks: Experimental evidence shows that WBP2 promotes anchorage-independent growth and invasiveness in breast epithelial cells .

• Drug Resistance: WBP2 has been implicated in mediating clinical drug resistance in breast cancer, although the detailed mechanisms require further investigation .

• miRNA Regulation: WBP2 is regulated by miRNAs such as miR-23a, miR-206, and miR-613 in breast cancer, and can also affect miRNA biogenesis through interactions with the microprocessor complex component DGCR8 .

These mechanisms collectively contribute to WBP2's role as an oncogene in breast cancer, making it a potential target for therapeutic interventions.

How does WBP2 function differ between breast cancer and lung cancer?

While WBP2 functions as an oncogene in both breast and lung cancers, there are notable differences in its mechanisms of action:

In lung cancer, WBP2 has been shown to work through a unique mechanism that is not directly dependent upon YAP. Instead, it competitively binds to the WW domain of WWC3 with LATS1 through its PPxY motifs, inhibiting the formation of the WWC3-LATS1 complex and ultimately suppressing the Hippo pathway to promote cancer progression .

What evidence exists for WBP2's involvement in other cancer types?

Beyond breast and lung cancers, there is emerging evidence for WBP2's involvement in other cancer types:

• Gliomas: Elevated WBP2 expression has been reported in human gliomas, where exogenous WBP2 increased cell proliferation, migration, and affected cell cycle progression .

• Hepatocellular Carcinoma (HCC): WBP2 expression is regulated by miR-485-5p in HCC, suggesting a potential role in liver cancer progression .

• Insulin Dysregulation-Associated Cancers: WBP2, along with USF-1, may act as causative factors in cancers associated with insulin dysregulation, such as pancreatic cancer, endometrial cancer, and certain forms of breast cancer .

The search results do not provide comprehensive information on WBP2's role in other cancer types, indicating an opportunity for further research to characterize its functions across a broader spectrum of malignancies.

What are the optimal methods for manipulating WBP2 expression in experimental models?

Researchers investigating WBP2 should consider several approaches for manipulating its expression:

• Overexpression Systems:

  • Plasmid-based overexpression using constitutive promoters (CMV, EF1α)

  • Inducible expression systems (Tet-On/Off)

  • Viral vectors (lentivirus, adenovirus) for difficult-to-transfect cells

  • CRISPR activation (CRISPRa) for endogenous gene upregulation

• Knockdown/Knockout Strategies:

  • siRNA for transient knockdown

  • shRNA for stable knockdown

  • CRISPR-Cas9 for complete knockout

  • Conditional knockout systems for temporal control

• Domain-Specific Manipulations:

  • Expression of mutated constructs targeting specific PY motifs

  • Domain deletion constructs

  • Point mutations at key phosphorylation or interaction sites

The choice of system depends on the specific research question and experimental model. For instance, studies in lung cancer have successfully employed gain- and loss-of-function experiments both in vitro and in vivo to assess WBP2's role in cancer progression . When targeting specific interactions, mutation of different PY motif sites has proven useful for distinguishing the relative specificity and binding affinity of different binding substrates .

How can researchers effectively analyze WBP2's impact on signaling pathways?

To analyze WBP2's impact on signaling pathways, researchers should implement a multi-faceted approach:

• Phosphorylation Analysis:

  • Western blotting with phospho-specific antibodies for key pathway components

  • Phosphoproteomics to identify global phosphorylation changes

  • Kinase activity assays

• Protein-Protein Interactions:

  • Co-immunoprecipitation followed by western blotting or mass spectrometry

  • Proximity ligation assays in situ

  • FRET/BRET for real-time interaction dynamics

  • Yeast two-hybrid or mammalian two-hybrid assays

• Transcriptional Output:

  • Luciferase reporter assays for specific pathway activities

  • ChIP-seq to identify genome-wide binding sites

  • RNA-seq for transcriptome-wide effects

  • qRT-PCR for validation of key target genes

• Subcellular Localization:

  • Immunofluorescence to track protein localization

  • Nuclear/cytoplasmic fractionation

  • Live-cell imaging with fluorescent fusion proteins

• Functional Readouts:

  • Proliferation assays

  • Migration/invasion assays

  • Apoptosis assays

  • In vivo tumor growth and metastasis assays

For example, in studying WBP2's role in the Hippo pathway in lung cancer, researchers determined that wild-type WBP2 could competitively bind to the WW domain of WWC3 with LATS1, reducing LATS1 phosphorylation and promoting YAP nuclear translocation . This was likely assessed through a combination of interaction studies, phosphorylation analyses, and localization experiments.

What are the challenges in developing WBP2-targeting therapeutic approaches?

Developing therapeutic approaches targeting WBP2 presents several challenges:

• Protein-Protein Interaction Targeting:

  • WBP2 functions through protein-protein interactions, which are traditionally difficult to target with small molecules

  • The PY motifs that mediate many of WBP2's interactions lack deep binding pockets that would facilitate drug development

  • Designing peptide mimetics or small molecules that specifically disrupt WBP2 interactions requires detailed structural understanding

• Pathway Redundancy and Compensation:

  • WBP2 integrates multiple signaling pathways; inhibiting one interaction might lead to compensation through other pathways

  • Understanding the complete interactome and its redundancies is necessary for effective targeting

• Context Dependency:

  • WBP2's functions differ between cancer types (e.g., breast vs. lung)

  • Therapeutic approaches may need to be tailored to specific cancer contexts

• Delivery and Specificity:

  • Ensuring that therapeutic agents reach the tumor site and enter cancer cells

  • Achieving specificity to avoid disrupting WBP2's normal physiological functions in hearing, neurological processes, endocrine function, etc.

• Biomarker Development:

  • Identifying patient subgroups most likely to benefit from WBP2-targeted therapies

  • Developing companion diagnostics to monitor treatment efficacy

To overcome these challenges, researchers might explore approaches such as structure-based drug design targeting specific interaction interfaces, development of proteolysis-targeting chimeras (PROTACs) to induce WBP2 degradation, or antisense oligonucleotides to reduce WBP2 expression in a tissue-specific manner.

How can WBP2 expression patterns be utilized for cancer diagnostics?

WBP2 expression patterns offer several potential applications in cancer diagnostics:

• Early Detection Biomarker:

  • Evidence supports WBP2 as a biomarker for early detection, particularly in breast cancer

  • Integration with other biomarkers may enhance sensitivity and specificity

• Prognostic Indicator:

  • In lung cancer, WBP2 expression is related to advanced pTNM stage, lymph node metastasis, and poor prognosis

  • Expression levels could help stratify patients by risk and inform treatment decisions

• Companion Diagnostics:

  • WBP2 status may predict response to targeted therapies

  • Could be particularly relevant for treatments targeting pathways where WBP2 plays a regulatory role

• Multi-omics Integration:

  • Combining WBP2 expression data with other molecular markers (mutations, epigenetic changes, etc.)

  • Pattern recognition across multiple markers may improve diagnostic accuracy

Implementation in clinical settings would require standardized detection methods, established cutoff values, and validation in large patient cohorts.

What is the potential for developing WBP2-targeted therapies?

WBP2-targeted therapies represent an emerging area with several potential approaches:

• Direct Targeting Strategies:

  • Small molecules or peptides disrupting specific WBP2 interactions

  • Proteolysis-targeting chimeras (PROTACs) to induce WBP2 degradation

  • Antisense oligonucleotides or RNAi-based approaches to reduce WBP2 expression

• Pathway-Based Approaches:

  • Targeting downstream effectors of WBP2-mediated signaling

  • Developing combination therapies targeting multiple nodes in WBP2-associated pathways

  • Synthetic lethality approaches exploiting dependencies created by WBP2 overexpression

• Context-Specific Applications:

  • In breast cancer, targeting WBP2's interactions with ER/PR or its role in drug resistance

  • In lung cancer, targeting the WBP2-WWC3-LATS1 regulatory axis

  • In insulin dysregulation-associated cancers, targeting the USF-1-WBP2 axis

As suggested in the search results, "blocking USF-1 and WBP2 loci may be a therapy for cancers associated with impaired insulin metabolism" . This highlights the potential for developing targeted approaches based on the specific mechanisms through which WBP2 contributes to different cancer types.

How might WBP2 research contribute to precision medicine approaches in oncology?

WBP2 research has significant potential to contribute to precision medicine in oncology:

• Patient Stratification:

  • WBP2 expression or activity could identify patient subgroups likely to benefit from specific treatments

  • Integration with other molecular markers to create comprehensive patient profiles

• Treatment Selection and Sequencing:

  • Informing the choice between available therapies based on WBP2 status

  • Guiding the sequence of multiple treatments for optimal outcomes

• Resistance Mechanisms:

  • Understanding WBP2's role in drug resistance, particularly in breast cancer

  • Developing strategies to overcome or prevent resistance

• Combination Therapies:

  • Rational design of combination approaches targeting WBP2 along with other cancer drivers

  • Identifying synergistic combinations to enhance efficacy while minimizing toxicity

• Monitoring Response:

  • Using WBP2 as a biomarker to monitor treatment efficacy

  • Early detection of resistance or relapse

The search results emphasize the vision for "new trends in WBP2 research in the space of molecular etiology of cancer, targeted therapeutics, and precision medicine" , highlighting the field's recognition of WBP2's potential in this area.

What are the most promising unexplored areas in WBP2 research?

Several promising unexplored or underdeveloped areas in WBP2 research include:

How might emerging technologies advance WBP2 research?

Emerging technologies have significant potential to advance WBP2 research:

• CRISPR-Based Technologies:

  • CRISPR screens to identify synthetic lethal partners of WBP2

  • Base editing for precise modification of WBP2 or its binding sites

  • CRISPRi/CRISPRa for temporal control of WBP2 expression

• Single-Cell Technologies:

  • Single-cell RNA-seq to characterize heterogeneity in WBP2 expression

  • Single-cell proteomics for protein-level analysis

  • Spatial transcriptomics to map WBP2 expression within the tumor microenvironment

• Advanced Imaging Techniques:

  • Super-resolution microscopy for detailed visualization of WBP2 interactions

  • Intravital imaging to track WBP2 dynamics in living organisms

  • Multiplexed imaging for simultaneous visualization of multiple pathway components

• Proteomics Innovations:

  • Proximity labeling techniques (BioID, APEX) to map WBP2's protein interaction network

  • Targeted proteomics for sensitive quantification of WBP2 and its binding partners

  • Advanced phosphoproteomics to characterize signaling dynamics

• Artificial Intelligence and Machine Learning:

  • Predictive modeling of WBP2-dependent processes

  • Drug discovery algorithms to identify WBP2-targeting compounds

  • Pattern recognition in multi-omics data sets including WBP2 status

• Organoid and 3D Culture Systems:

  • Patient-derived organoids to study WBP2 in more physiologically relevant contexts

  • 3D co-culture systems to examine WBP2's role in tumor-stroma interactions

What interdisciplinary approaches might enhance our understanding of WBP2 biology?

Interdisciplinary approaches hold significant promise for advancing WBP2 research:

• Chemical Biology + Structural Biology:

  • Development of chemical probes to selectively modulate WBP2 functions

  • Structure-based design of inhibitors targeting specific WBP2 interactions

• Systems Biology + Computational Modeling:

  • Integration of multiple data types to model WBP2's position in cellular networks

  • Simulation of pathway perturbations to predict therapeutic opportunities

• Immunology + Cancer Biology:

  • Investigation of WBP2's role in cancer-immune interactions

  • Development of immunotherapeutic approaches targeting WBP2-expressing tumors

• Clinical Oncology + Molecular Diagnostics:

  • Validation of WBP2 as a biomarker in diverse patient populations

  • Implementation of WBP2 testing in clinical trials and practice

• Developmental Biology + Cancer Biology:

  • Comparative analysis of WBP2's roles in development and cancer

  • Insights from WBP2's functions in hearing and neurological processes

• Bioinformatics + Translational Research:

  • Mining of public databases to identify correlations between WBP2 and clinical outcomes

  • Integration of genomic, transcriptomic, and proteomic data to build comprehensive models

• Medicinal Chemistry + Biology:

  • Design and development of small molecules targeting WBP2 or its interactions

  • Optimization of lead compounds for potential therapeutic applications

These interdisciplinary approaches would help address the complex biology of WBP2 from multiple perspectives, potentially leading to more comprehensive understanding and more effective therapeutic strategies.

What are common challenges in detecting and measuring WBP2 expression?

Researchers studying WBP2 may encounter several challenges in detection and measurement:

• Antibody Specificity and Sensitivity:

  • Commercial antibodies may vary in quality and specificity

  • Cross-reactivity with related proteins (e.g., WBP2NL) can confound results

  • Recommendation: Validate antibodies using positive and negative controls, including WBP2 knockout samples

• Post-Translational Modifications:

  • Phosphorylation or other modifications may mask epitopes

  • Different detection methods may be biased toward specific protein states

  • Recommendation: Use multiple antibodies targeting different epitopes and consider phospho-specific antibodies when appropriate

• Low Expression Levels:

  • WBP2 may be expressed at low levels in certain tissues or cell types

  • Standard detection methods may lack sufficient sensitivity

  • Recommendation: Consider amplification steps, more sensitive detection methods, or enrichment procedures

• Subcellular Localization:

  • WBP2 may shuttle between cellular compartments, affecting detection

  • Different fixation methods may alter localization patterns

  • Recommendation: Use fractionation approaches and compare multiple fixation/permeabilization protocols

• Splice Variants and Isoforms:

  • Potential existence of multiple WBP2 isoforms with different properties

  • Primers or antibodies may not detect all relevant forms

  • Recommendation: Design detection strategies targeting conserved regions or use multiple approaches targeting different regions

To address these challenges, researchers should implement rigorous validation procedures and consider using complementary detection methods to corroborate findings.

How can researchers resolve conflicting data regarding WBP2 function?

When faced with conflicting data about WBP2 function, researchers should consider several strategies:

• Context Dependency Analysis:

  • Systematically compare experimental conditions (cell types, cancer types, etc.)

  • Determine whether conflicts might reflect genuine biological differences in different contexts

  • Test the same hypothesis across multiple model systems

• Methodological Evaluation:

  • Compare technical approaches used in conflicting studies

  • Assess potential biases or limitations in different methods

  • Implement multiple independent techniques to address the same question

• Temporal Considerations:

  • Investigate whether conflicts might reflect time-dependent effects

  • Conduct time-course experiments to capture dynamic processes

  • Consider acute versus chronic manipulations of WBP2 levels

• Dose-Dependent Effects:

  • Test whether conflicting results might reflect differences in WBP2 expression levels

  • Implement titratable expression systems to examine dose-response relationships

  • Consider potential threshold effects

• Pathway Cross-Talk:

  • Examine the status of interacting pathways in different experimental systems

  • Test whether pathway activities modulate WBP2 function

  • Investigate compensatory mechanisms that might mask phenotypes

• Direct Replication Studies:

  • Attempt to directly replicate conflicting findings using identical conditions

  • Collaborate with laboratories reporting conflicting results

  • Consider multi-laboratory collaborative studies for key controversies

Researchers should maintain transparency about conflicting data in publications and work toward resolving contradictions rather than selectively reporting compatible findings.

What experimental controls are critical for WBP2 functional studies?

Robust experimental controls are essential for reliable WBP2 functional studies:

• Expression Controls:

  • Verification of WBP2 overexpression or knockdown/knockout efficiency

  • Use of multiple independent clones or populations

  • Rescue experiments to confirm specificity of phenotypes

• Domain-Specific Controls:

  • Function-disrupting mutations (e.g., in PY motifs)

  • Domain deletion constructs

  • Wild-type controls expressed at comparable levels

• Pathway Activity Controls:

  • Monitoring the status of pathways regulated by or regulating WBP2

  • Positive and negative controls for pathway activation/inhibition

  • Time-course analyses to capture dynamic responses

• Cell Type Controls:

  • Comparison across multiple cell lines or primary cells

  • Matched normal and cancer cells when relevant

  • Isogenic cell lines differing only in WBP2 status

• Technical Controls:

  • Vehicle controls for all treatments

  • Non-targeting siRNA/shRNA controls

  • Empty vector controls for expression constructs

  • Isotype controls for antibodies

• Functional Assay Controls:

  • Positive and negative controls for each functional assay

  • Dose-response curves for pharmacological interventions

  • Time-course analyses to establish optimal experimental windows

Implementing these controls ensures that observed effects can be confidently attributed to WBP2 rather than experimental artifacts or off-target effects, and enables meaningful interpretation of results across different experimental settings.