wdr4 Antibody

What is WDR4 and why is it significant in research?

WDR4 (WD repeat domain 4) belongs to the WD repeat family of proteins, characterized by the presence of WD-repeats that form propeller-like tertiary structures. WDR4 forms a complex with METTL1 (methyltransferase like 1), which is essential for the 7-methylguanosine (m7G) modification of tRNA, a vital modification for proper translation and gene expression . WDR4 has emerged as a significant research target due to its involvement in:

• Cancer progression and metastasis in hepatocellular carcinoma and bladder cancer

• Neural development, where mutations are associated with primordial dwarfism and microcephaly

• Cellular processes including cell cycle progression, signal transduction, and RNA processing

What types of WDR4 antibodies are currently available for research applications?

Several validated WDR4 antibodies are currently available for research use:

The choice of antibody should be based on your specific application and species of interest .

What are the recommended applications for WDR4 antibodies?

WDR4 antibodies have been validated for various research applications:

• Western Blotting (WB): The most common application, typically using 1:1000 dilution. WDR4 is typically detected at 30-45 kDa depending on the isoform .

• Immunoprecipitation (IP): Useful for studying protein-protein interactions, such as WDR4-METTL1 or WDR4-DDX20 complexes .

• Immunofluorescence (IF)/Immunocytochemistry (ICC): For detecting subcellular localization of WDR4, which can vary between cytoplasmic and nuclear depending on cellular context .

• Immunohistochemistry with paraffin-embedded sections (IHC-P): For analyzing WDR4 expression in tissue samples, particularly in cancer progression studies .

• ELISA: For quantitative detection of WDR4 protein levels .

How should WDR4 antibody be validated for cancer research applications?

For cancer research applications, comprehensive validation of WDR4 antibodies should include:

• Specificity testing: Use positive controls (tissues or cell lines with known WDR4 overexpression such as Hep-3B, Hep-G2, HCC-LM3, and Huh-7 for hepatocellular carcinoma) and negative controls (WDR4 knockdown cells) .

• Cross-validation with multiple techniques: Compare protein detection using at least two different antibodies or detection methods (e.g., mass spectrometry) .

• Expression correlation: Confirm that protein levels detected by the antibody correlate with mRNA expression data from qRT-PCR .

• Isoform specificity: WDR4 exists in three isoforms (isoforms 1 and 2 are 45kDa, isoform 3 is 30kDa); ensure the antibody detects the relevant isoform for your research .

• Functional validation: Confirm that phenotypic changes observed after WDR4 knockdown or overexpression are consistent with the literature (e.g., changes in cell proliferation, migration, or invasion) .

What are the optimal tissue processing methods for WDR4 immunohistochemistry in clinical samples?

For optimal WDR4 immunohistochemical staining in clinical samples:

• Tissue fixation: Use 10% neutral-buffered formalin fixation for 24-48 hours to preserve antigen integrity.

• Antigen retrieval: Heat-induced epitope retrieval (HIER) in citrate buffer (pH 6.0) or EDTA buffer (pH 9.0) is recommended. For WDR4, citrate buffer has shown reliable results in hepatocellular carcinoma and bladder cancer tissues .

• Blocking and antibody dilution: Use 3-5% BSA or normal serum for blocking non-specific binding. Antibody dilutions typically range from 1:100 to 1:500 for IHC-P applications.

• Quantification: Use a standardized scoring system such as the H-score (combines intensity and percentage of positive cells) or immunoreactive score (IRS) to quantify WDR4 expression. This is particularly important for correlation with clinicopathological features such as tumor grade, stage, and patient survival .

• Controls: Include both positive controls (e.g., HCC or bladder cancer tissue with known WDR4 overexpression) and negative controls (normal adjacent tissue or antibody diluent only) .

How can researchers effectively study WDR4-protein interactions using immunoprecipitation?

To effectively study WDR4-protein interactions:

• Co-immunoprecipitation (co-IP) protocol:

  • Use mild lysis buffers (containing 0.5-1% NP-40 or Triton X-100) to preserve protein-protein interactions

  • Pre-clear lysates with protein A/G beads to reduce non-specific binding

  • Incubate with WDR4 antibody (typical ratio: 1:100) overnight at 4°C

  • Capture antibody-protein complexes with protein A/G beads

  • Wash stringently (at least 3-5 times) to remove non-specific interactions

  • Elute and analyze by Western blotting for interacting partners

• Known interaction partners to validate:

  • METTL1 (primary binding partner in tRNA modification)

  • DDX20 (in bladder cancer progression)

  • Egr1 (in transcriptional regulation)

  • EIF2A (in hepatocellular carcinoma)

• Mass spectrometry analysis: For unbiased discovery of novel interaction partners, as demonstrated in bladder cancer studies where co-IP followed by LC-MS/MS identified 251 and 281 potential WDR4-interacting proteins in UM-UC-3 and 5637 cells, respectively .

How can researchers differentiate between the roles of WDR4 in cancer progression versus neurodevelopmental disorders?

Differentiating between WDR4's roles in cancer and neurodevelopmental contexts requires specific experimental approaches:

• Mutation analysis:

  • In neurodevelopmental disorders: Focus on loss-of-function mutations that impair tRNA methylation (usually point mutations affecting WDR4-METTL1 interaction)

  • In cancer: Examine expression levels rather than mutations, as overexpression is the predominant mechanism

• Functional readouts:

  • For neurodevelopmental studies: Measure m7G tRNA modification levels, cilia formation, protein synthesis rates, and proteasomal activity

  • For cancer studies: Assess cell proliferation, migration, invasion, and resistance to therapeutics like sorafenib

• Downstream pathway analysis:

  • In neurodevelopment: Focus on mTOR pathway components, which are affected by impaired tRNA m7G modification, and endoplasmic reticulum stress markers

  • In cancer: Examine cell cycle regulators (CDC25C, CCNB1, P21), EMT markers (E-cadherin, N-cadherin, vimentin), and ARRB2 expression

• Model systems:

  • For neurodevelopmental research: Use patient-derived iPSCs differentiated into neural stem cells, or zebrafish embryos harboring WDR4 mutations

  • For cancer research: Utilize established cancer cell lines (UM-UC-3, 5637 for bladder cancer; Hep-3B, Hep-G2, HCC-LM3, and Huh-7 for HCC) and in vivo metastasis models

What methodological approaches can resolve contradictory findings regarding WDR4's subcellular localization?

To address contradictory findings regarding WDR4's subcellular localization:

• Multiple detection methods:

  • Combine immunofluorescence with subcellular fractionation followed by Western blotting

  • Use confocal microscopy with z-stack imaging to precisely determine localization

  • Consider live-cell imaging with fluorescently tagged WDR4 to monitor dynamic localization changes

• Context-specific analysis:

  • Evaluate localization under different cellular conditions (e.g., cell cycle stages, stress conditions)

  • Compare localization in normal versus cancer cells, as WDR4 may shuttle between the cytoplasm and nucleus in a context-dependent manner

  • Assess localization in the presence or absence of binding partners like METTL1 or DDX20

• Isoform-specific detection:

  • Use antibodies specific for different WDR4 isoforms, as different isoforms may localize differently

  • Perform isoform-specific knockdown/overexpression experiments to determine differential localization

• Functional validation:

  • Correlate subcellular localization with functionality (e.g., using tRNA methylation assays)

  • Use nuclear export/import inhibitors to confirm shuttling capability

How can researchers effectively measure and interpret changes in tRNA m7G modification levels in WDR4 functional studies?

To effectively measure tRNA m7G modification levels:

• Analytical methods:

  • HPLC-MS/MS analysis: Most accurate method for quantifying m7G levels in purified tRNA

  • m7G-specific antibody-based methods: Such as m7G-MeRIP-seq for transcriptome-wide profiling

  • Thin layer chromatography (TLC): For relative quantification of modified nucleosides

• Experimental design considerations:

  • Include both positive controls (wild-type cells) and negative controls (METTL1 knockout or WDR4 knockout cells)

  • Analyze specific tRNA species known to be m7G-modified (typically at position 46)

  • Account for total tRNA levels, as changes in modification could reflect changes in tRNA abundance

• Functional correlation:

  • Link m7G levels to translation efficiency using polysome profiling or ribosome footprinting

  • Correlate with phenotypic outcomes (e.g., cell proliferation, neural development)

  • Assess impact on stress response pathways, particularly relevant in neurodevelopmental contexts

• Rescue experiments:

  • Overexpress wild-type WDR4 in mutant cells to confirm that phenotypic rescue correlates with restoration of m7G levels

  • Use patient-derived WDR4 mutations to establish causality between specific mutations, m7G levels, and phenotypes

What are the common issues with WDR4 antibody specificity and how can researchers address them?

Common specificity issues with WDR4 antibodies include:

• Multiple bands in Western blots:

  • Cause: Detection of multiple isoforms (30kDa and 45kDa variants), non-specific binding, or post-translational modifications

  • Solution: Validate with positive controls (WDR4 overexpression) and negative controls (WDR4 knockdown); use antigen pre-absorption tests; optimize antibody concentration and blocking conditions

• Weak or inconsistent signal:

  • Cause: Low WDR4 expression in certain cell types, poor antibody sensitivity, or protein degradation

  • Solution: Optimize protein extraction methods (use protease inhibitors); increase antibody concentration or incubation time; enhance signal with amplification systems

• High background:

  • Cause: Non-specific binding, insufficient blocking, or too high antibody concentration

  • Solution: Optimize blocking (try different blocking agents like 5% BSA or 5% milk); increase washing steps; reduce antibody concentration; use monoclonal antibodies for higher specificity

• Cross-reactivity with related proteins:

  • Cause: WDR4 is a member of the WD-repeat family with structural similarity to other family members

  • Solution: Use antibodies raised against unique regions of WDR4; validate with recombinant proteins of related family members; perform immunoprecipitation followed by mass spectrometry to confirm specificity

How can researchers optimize WDR4 knockdown and overexpression systems for functional studies?

For optimal WDR4 knockdown and overexpression systems:

• Knockdown strategies:

  • siRNA: Use at least 3 independent siRNAs targeting different regions of WDR4 mRNA to confirm specificity of phenotypes

  • shRNA: For stable knockdown, target conserved regions across isoforms; validate knockdown efficiency by both qRT-PCR and Western blot

  • CRISPR-Cas9: Design guides with minimal off-target effects; create heterozygous and homozygous knockout lines to assess dose-dependent effects

• Overexpression systems:

  • Use tagged constructs (HA, FLAG, or GFP) for easy detection and immunoprecipitation

  • Consider inducible systems (e.g., Tet-On) to control expression levels and timing

  • Validate correct subcellular localization of overexpressed protein compared to endogenous WDR4

• Controls and validation:

  • Include appropriate controls (empty vector, scrambled siRNA)

  • Validate expression changes at both mRNA and protein levels

  • Test for off-target effects by rescuing the phenotype with an siRNA-resistant WDR4 construct

• Phenotypic analysis:

  • For cancer studies: Assess proliferation (Ki67 staining, EdU incorporation), cell cycle progression (PI staining), migration/invasion (wound healing, transwell assays), and apoptosis (Annexin V, TUNEL)

  • For neurodevelopmental studies: Examine cilia formation, proteasome activity, protein synthesis rates, and neural differentiation

What methodology should be employed to investigate the relationship between WDR4 and m7G tRNA modification in different experimental contexts?

To investigate WDR4-mediated m7G tRNA modification:

• Techniques for m7G detection:

  • LC-MS/MS: Gold standard for direct quantification of m7G modification

  • m7G-MeRIP-seq: For transcriptome-wide mapping of m7G modifications

  • m7G-specific antibodies: For immunofluorescence or dot blot analysis

  • Northern blotting with m7G-specific probes: For analyzing specific tRNA species

• Experimental design:

  • Compare m7G levels in WDR4 wild-type, knockdown, knockout, and rescue conditions

  • Analyze m7G modification in the context of METTL1 manipulation, as they function together

  • Perform structure-function studies with WDR4 mutants to identify domains critical for m7G formation

• Functional consequences assessment:

  • Polysome profiling: To assess impact on translation efficiency

  • Ribosome footprinting: To identify specific mRNAs affected by altered m7G levels

  • Pulse-chase labeling: To measure global protein synthesis rates

• Disease-relevant models:

  • Use patient-derived mutations for structure-function studies

  • Develop cell and animal models that recapitulate disease-associated WDR4 mutations

  • Test therapeutic strategies that aim to restore m7G levels or mitigate downstream consequences, such as TUDCA treatment to reduce endoplasmic reticulum stress or AAV-mediated WDR4 restoration

How might WDR4 function as a therapeutic target in cancer, and what experimental approaches could validate this potential?

To evaluate WDR4 as a therapeutic target in cancer:

• Target validation approaches:

  • Use conditional knockdown systems in established tumors to confirm that WDR4 inhibition leads to tumor regression

  • Assess synthetic lethality by combining WDR4 inhibition with standard therapies (e.g., sorafenib for HCC)

  • Evaluate effects on cancer stem cells and tumor-initiating capacity using limiting dilution assays

• Drug development strategies:

  • Structure-based drug design targeting the WDR4-METTL1 interface

  • Small molecule screening against the WD-repeat domain

  • Peptide inhibitors that disrupt specific protein-protein interactions

  • Evaluate repurposing of existing drugs that may affect WDR4 function or expression

• Efficacy and specificity testing:

  • In vitro models: Use 2D and 3D culture systems with multiple cancer cell lines

  • In vivo models: Xenograft models, including the popliteal lymph node metastasis model for bladder cancer

  • Patient-derived xenografts to assess heterogeneity in response

  • Combination therapy approaches with established agents

• Biomarker development:

  • Develop IHC-based protocols for stratifying patients based on WDR4 expression levels

  • Identify gene signatures associated with WDR4 dependency

  • Establish liquid biopsy approaches to monitor treatment response

What methodological approaches can bridge the gap between WDR4's roles in cancer and neurodevelopmental disorders?

To bridge WDR4 research across disease contexts:

• Comparative molecular profiling:

  • Perform transcriptome and proteome analysis in both neuronal and cancer models with WDR4 manipulation

  • Identify common and distinct pathways affected by WDR4 alteration

  • Compare m7G modification landscapes across cell types

• Unified experimental systems:

  • Develop isogenic cell line panels with either WDR4 overexpression (cancer model) or patient-derived mutations (neurodevelopmental model)

  • Use inducible systems to study dose-dependent effects across cell types

  • Perform cross-disease rescue experiments (Can cancer-associated WDR4 overexpression rescue neurodevelopmental phenotypes?)

• Translational research approaches:

  • Analyze WDR4 expression in neurodevelopmental disorder patient samples

  • Evaluate neurological phenotypes in cancer patients with WDR4-overexpressing tumors

  • Develop therapeutic approaches that could benefit both conditions (e.g., targeting downstream pathways rather than WDR4 directly)

• Advanced model systems:

  • Develop organoid models for both cancer and neurodevelopment

  • Use CRISPR-engineered animal models with tissue-specific WDR4 alterations

  • Implement patient-derived iPSCs differentiated into relevant cell types for both disease contexts

How can researchers effectively study the complex relationship between WDR4, protein homeostasis, and cilia formation in the context of neurodevelopmental disorders?

To investigate WDR4's role in protein homeostasis and cilia formation:

• Integrated analytical approaches:

  • Combine live-cell imaging of cilia formation with quantitative proteomics

  • Monitor proteasome activity and ubiquitin pools in real-time using fluorescent reporters

  • Perform pulse-chase experiments to assess protein synthesis and degradation rates

• Mechanistic studies:

  • Use ubiquitin chain-specific antibodies to characterize changes in ubiquitination patterns

  • Investigate the ubiquitin-proteasome system in WDR4-deficient cells

  • Assess protein folding and ER stress markers in relation to cilia defects

  • Perform epistasis experiments with proteasome inhibitors and free ubiquitin supplementation

• Therapeutic testing platforms:

  • Screen compounds that restore cilia formation in WDR4-deficient cells

  • Test proteasome modulators and chaperone inducers for their ability to rescue phenotypes

  • Evaluate TUDCA and similar compounds that reduce ER stress in various model systems

• Translational approaches:

  • Develop quantitative assays for cilia defects that could serve as biomarkers

  • Establish in vitro diagnostics to predict patient-specific responses to potential therapeutics

  • Create patient stratification strategies based on molecular phenotypes