wdr75 Antibody

What is WDR75 and why is it important for cellular function?

WDR75 (WD Repeat Domain 75) is a nucleolar protein that plays a crucial role in ribosome biogenesis. It functions as a ribosome biogenesis factor and is part of the small subunit (SSU) processome, which is the first precursor of the small eukaryotic ribosomal subunit. WDR75 is involved in:

• Nucleolar processing of pre-18S ribosomal RNA

• Positive regulation of transcription by RNA polymerase I

• Supporting pre-rRNA transcription by maintaining optimal levels of RPA194 (a key subunit of RNA polymerase I)

The importance of WDR75 is underscored by the fact that its depletion activates the RPL5/RPL11-dependent p53 stabilization checkpoint, leading to impaired cellular proliferation and senescence, highlighting its essential role in normal cellular function .

Which applications are WDR75 antibodies validated for?

Based on manufacturer data, commercially available WDR75 antibodies have been validated for multiple applications:

The most robust validation has been performed for IHC and ICC/IF applications, with detailed subcellular localization data available through resources like the Human Protein Atlas .

What is the typical subcellular localization pattern for WDR75 when detected by antibodies?

WDR75 antibodies typically detect a predominantly nucleolar localization pattern in immunofluorescence studies. This observation has been confirmed through multiple approaches:

• GFP-tagged WDR75 localizes predominantly to nucleoli under unperturbed growth conditions

• Subcellular fractionation followed by immunoblotting validates the nucleolar localization of endogenous WDR75

• Immunofluorescence staining with WDR75 antibodies shows localization to the nucleoplasm and nucleoli

Interestingly, under ribosomal stress conditions (e.g., treatment with Actinomycin D or BMH-21), WDR75 redistributes to nucleolar caps, colocalizing with markers like fibrillarin (FIB) and RPA194 .

What criteria should I consider when selecting a WDR75 antibody for my research?

When selecting a WDR75 antibody, consider these key factors:

• Epitope location: Some antibodies target the N-terminal region , while others target specific internal sequences . The epitope location matters particularly if you're studying:

  • Specific protein domains (WDR75 contains 13 WD40 repeats)

  • Protein-protein interactions (e.g., with UTP4 or UTP18)

  • Transcript variants with different start codons

• Host species and clonality: Most available WDR75 antibodies are rabbit polyclonals , but consider:

  • Cross-reactivity concerns if working with rabbit tissues

  • Batch-to-batch variability with polyclonals

  • Compatibility with other antibodies in multi-labeling experiments

• Validated applications: Ensure the antibody is validated for your specific application with data demonstrating:

  • Appropriate subcellular localization (nucleolar pattern)

  • Expected molecular weight (~90kDa)

  • Specificity through knockdown controls

• Species reactivity: Most WDR75 antibodies react with human samples, with some cross-reactivity to mouse and rat (88% sequence identity) .

How should I optimize immunofluorescence protocols for detecting WDR75 in the nucleolus?

Detecting nucleolar proteins like WDR75 requires special considerations in immunofluorescence protocols:

• Fixation optimization:

  • For nucleolar proteins, paraformaldehyde fixation (4%, 10-15 minutes) preserves nucleolar structure

  • Methanol fixation may improve accessibility to some nucleolar epitopes but can disrupt certain protein-protein interactions

• Permeabilization considerations:

  • Use 0.1-0.5% Triton X-100 for adequate nuclear permeabilization

  • For dense nucleolar structures, consider extending permeabilization time

  • Alternative: 0.5% Saponin may provide gentler permeabilization while maintaining nucleolar morphology

• Antibody concentration optimization:

  • Start with manufacturer's recommended range (0.25-2 μg/mL)

  • Titrate to minimize background while maintaining specific nucleolar signal

  • Include controls: (a) secondary-only control, (b) known nucleolar marker (fibrillarin)

• Co-staining strategy:

  • Include nucleolar markers for colocalization (fibrillarin, RPA194)

  • Consider DAPI staining to visualize nuclear morphology

  • For studying stress responses, include markers of nucleolar caps to confirm WDR75 redistribution under stress conditions

• Signal amplification options:

  • For weak signals, consider tyramide signal amplification

  • Use of confocal microscopy with z-stacking to properly resolve nucleolar structures

What controls should be included when using WDR75 antibodies in experimental procedures?

Proper experimental controls are essential for interpreting results with WDR75 antibodies:

The rescue control is particularly important, as demonstrated in previous research where ectopically expressed WDR75-GFP (resistant to siRNA targeting the 3′UTR) functionally rescued the p53-triggering phenotype of WDR75 depletion, confirming specificity of the observed effects .

How can WDR75 antibodies be used to study ribosome biogenesis stress responses?

WDR75 antibodies provide powerful tools for investigating ribosome biogenesis stress responses:

• Nucleolar reorganization studies:

  • Under ribotoxic stress (Actinomycin D or BMH-21 treatment), WDR75 redistributes to nucleolar caps

  • WDR75 antibodies can track this redistribution through time-course experiments

  • Co-staining with other markers (fibrillarin, RPA194) characterizes comprehensive nucleolar reorganization

• p53 checkpoint activation analysis:

  • WDR75 depletion activates the p53-p21 axis

  • Combined staining for WDR75, p53, and p21 can assess the relationship between WDR75 levels and checkpoint activation

  • Phospho-specific antibodies against p53 can distinguish between DNA damage-induced and ribosome stress-induced p53 activation

• Ribosomal protein interaction studies:

  • IP with WDR75 antibodies followed by mass spectrometry can identify interaction partners

  • Co-IP experiments can verify specific interactions with RPL5/RPL11 during stress responses

  • Proximity ligation assays can visualize dynamic interactions between WDR75 and other SSU processome components

• Cell cycle analysis integration:

  • Combined staining for WDR75, EdU incorporation, and cell cycle markers

  • Quantitative image analysis correlating WDR75 levels with proliferation indicators

  • Flow cytometry sorting based on WDR75 levels followed by cell cycle analysis

What methodological approaches can address the challenges of studying WDR75 transcript variants with antibodies?

Studying WDR75 transcript variants presents unique challenges requiring specialized methodological approaches:

• Transcript variant-specific detection strategies:

  • Standard WDR75 antibodies may not distinguish between transcript variants

  • Design custom antibodies against unique N-terminal sequences in transcript variants

  • For the human and bonobo transcript variants (X2) that start 64 amino acids downstream, develop antibodies against the unique first seven amino acids

• Integrated RNA-protein analysis approaches:

  • Combine RNA-seq to quantify transcript variant expression with protein-level detection

  • RT-PCR with variant-specific primers followed by Western blotting

  • Single-cell analysis correlating transcript variant expression with protein localization

• Structural impact assessment:

  • Use 3D structural modeling to predict impacts of variants on protein folding

  • The transcript variants fold into 54 beta strands (three fewer than reference sequences)

  • Design experiments to test functional implications of these structural differences

• Evolution-informed analysis:

  • Consider the impact of evolutionary conservation on epitope accessibility

  • Sites under purifying selection (25% of WDR75 sites) may affect antibody binding

  • Beta-sheet regions show ~5x stronger purifying selection than coil regions, potentially affecting epitope exposure

How can WDR75 antibodies be used to investigate the mechanistic relationship between WDR75 and RPA194?

Research has shown that WDR75 positively affects pre-rRNA synthesis by stabilizing RPA194. WDR75 antibodies can help elucidate this mechanism through:

• Proximity-based interaction studies:

  • Proximity ligation assays (PLA) using antibodies against WDR75 and RPA194

  • FRET/FLIM approaches with labeled antibodies to assess direct interactions

  • IP-mass spectrometry to identify bridging proteins in the WDR75-RPA194 complex

• Quantitative correlation analysis:

  • Dual immunofluorescence with WDR75 and RPA194 antibodies

  • Single-cell quantification of intensity correlation

  • Time-course analysis after WDR75 depletion to determine kinetics of RPA194 reduction

• Domain-specific interaction mapping:

  • Competition assays with peptides corresponding to WDR75 domains

  • Structure-guided mutagenesis followed by co-IP with WDR75 and RPA194 antibodies

  • Selective domain deletion constructs to identify regions required for RPA194 stabilization

• Functional recovery experiments:

  • WDR75 depletion reduces RPA194 levels and pre-rRNA synthesis

  • Rescue experiments with WDR75 mutants lacking specific WD40 repeats

  • Assessment of domain contributions to RPA194 stabilization and pre-rRNA transcription

How can I address the issue of cross-reactivity when using WDR75 antibodies?

Cross-reactivity is a common challenge with antibodies against conserved proteins like WDR75:

• Sources of WDR75 antibody cross-reactivity:

  • High conservation across mammals (e.g., 88% sequence identity between human and mouse/rat)

  • Similar WD40 repeat domains in related proteins

  • Non-specific binding to other nucleolar proteins

• Validation approaches for confirming specificity:

  • siRNA knockdown of WDR75 in Western blot and immunofluorescence

  • Pre-absorption with immunizing peptide to confirm epitope specificity

  • Testing antibody on WDR75 knockout cell lines (if available)

  • Comparison of staining patterns across multiple WDR75 antibodies targeting different epitopes

• Optimization strategies to minimize cross-reactivity:

  • Increase blocking time and concentration (5% BSA or 5-10% normal serum)

  • Include 0.1-0.3% Triton X-100 in antibody diluent to reduce non-specific binding

  • Optimize antibody concentration and incubation time

  • Consider using monoclonal antibodies for higher specificity

  • For tissues with high background, use biotin-free detection systems

The specificity of WDR75 antibodies should be validated in each experimental system, as documented in previous studies where siRNA knockdown confirmed antibody specificity .

What approaches can resolve contradictory results when studying WDR75 using different antibodies?

When different WDR75 antibodies yield contradictory results, consider these systematic approaches:

• Epitope mapping analysis:

  • Compare epitope locations of different antibodies

  • N-terminal antibodies may give different results than those targeting internal regions

  • Potential post-translational modifications may block epitope accessibility

  • Protein-protein interactions may mask certain epitopes

• Expression system variables:

  • Recombinant versus endogenous WDR75 detection may differ

  • GFP-tagged WDR75 localization has been validated against endogenous protein

  • Consider effects of overexpression on localization and interactions

• Methodological cross-validation:

  • Combine multiple detection methods (WB, IF, IP)

  • Use fractionation approaches to confirm subcellular localization

  • Consider non-antibody methods (RNA-seq, mass spectrometry)

  • Employ CRISPR tagging of endogenous WDR75 for validation

• Experimental condition variations:

  • Cell cycle stage affects nucleolar morphology

  • Stress responses redistribute WDR75 to nucleolar caps

  • Fixation methods significantly impact nucleolar protein detection

  • Cell confluency affects ribosome biogenesis and nucleolar structure

• Systematic antibody comparison:

  • Side-by-side testing under identical conditions

  • Titration series for each antibody

  • Detailed documentation of buffer compositions and incubation parameters

  • Blind analysis of staining patterns to avoid confirmation bias

What advanced imaging techniques can improve detection of WDR75 in nucleolar structures?

Studying nucleolar proteins like WDR75 benefits from specialized imaging approaches:

• Super-resolution microscopy options:

  • Structured illumination microscopy (SIM) provides 2x resolution improvement

  • Stimulated emission depletion (STED) microscopy can resolve nucleolar subcompartments

  • Single-molecule localization microscopy (PALM/STORM) for nanoscale distribution analysis

  • Expansion microscopy physically enlarges samples for enhanced resolution

• Live-cell imaging strategies:

  • Fluorescent protein tagging (as validated with WDR75-GFP)

  • HaloTag or SNAP-tag systems for pulse-chase experiments

  • Lattice light-sheet microscopy for 3D dynamics with reduced phototoxicity

  • Photoactivatable fluorophores for precise tracking of nucleolar reorganization

• Correlative light and electron microscopy (CLEM):

  • Combines immunofluorescence localization with ultrastructural context

  • immunogold labeling for electron microscopy

  • Cryo-electron tomography for 3D ultrastructural analysis

• Quantitative image analysis approaches:

  • 3D reconstruction of nucleolar structures

  • Colocalization analysis using Pearson's or Manders' coefficients

  • FRAP (fluorescence recovery after photobleaching) to study dynamics

  • Single-particle tracking for movement within nucleolar compartments

• Multi-modal imaging integration:

  • Combined detection of WDR75 protein (antibody) and pre-rRNA (FISH)

  • Metabolic labeling of nascent RNA with EU combined with WDR75 immunofluorescence

  • Multi-spectral imaging for simultaneous detection of multiple nucleolar components

  • Label-free techniques (Raman microscopy) combined with immunofluorescence

How might WDR75 antibodies contribute to understanding ribosomopathies and cancer?

WDR75 antibodies offer promising tools for investigating the role of ribosome biogenesis in disease:

• Ribosomopathy diagnostic applications:

  • WDR75 antibodies could potentially serve as diagnostic tools for ribosomopathies

  • Altered WDR75 expression/localization patterns may correlate with disease subtypes

  • Integration with other ribosomal protein markers for comprehensive profiling

• Cancer biomarker potential:

  • Aberrantly high levels of WDR75 mRNA have been identified as an unfavorable prognostic marker for renal and liver cancer

  • WDR75 antibodies could help validate protein-level correlations with prognosis

  • Tissue microarray studies across cancer types to assess broader relevance

• Therapeutic target validation:

  • The p53-dependent cell cycle arrest upon WDR75 knockdown suggests potential therapeutic relevance

  • Antibodies can help validate WDR75 as a potential cancer target

  • Correlation of WDR75 levels with sensitivity to ribosome biogenesis inhibitors

• Mechanistic studies of oncogenic stress:

  • Investigation of WDR75's role in nucleolar stress response in cancer cells

  • Analysis of WDR75-dependent p53 checkpoint in different genetic backgrounds

  • Correlation of WDR75 expression with cancer cell sensitivity to nucleolar stress inducers

What methodological approaches can integrate WDR75 antibody-based detection with functional genomics and proteomics?

Modern research benefits from integrating antibody-based detection with multi-omics approaches:

• Integrative ChIP-seq approaches:

  • ChIP-seq using WDR75 antibodies to identify genomic binding sites

  • Integration with RNA Pol I ChIP-seq data to elucidate cooperative binding

  • Correlation with pre-rRNA transcription sites

  • Analysis of chromatin states at WDR75 binding regions

• Spatial proteomics applications:

  • Proximity labeling (BioID, APEX) with WDR75 to identify proximal proteins in different cellular states

  • Mass spectrometry combined with WDR75 IP to identify interaction partners

  • Spatial proteomics mapping of nucleolar reorganization during stress

  • Cross-linking mass spectrometry to identify direct interaction interfaces

• Single-cell multi-omics integration:

  • Single-cell IF for WDR75 combined with scRNA-seq

  • Correlation of WDR75 protein levels with transcriptomic signatures

  • CITE-seq adaptation for simultaneous protein and RNA profiling

  • Spatial transcriptomics to correlate WDR75 localization with local RNA processing

• CRISPR screening approaches:

  • CRISPR screens for genes affecting WDR75 localization/function

  • Antibody-based readouts for high-content CRISPR screens

  • CRISPR activation/inhibition of WDR75 followed by proteomic analysis

  • Synthetic lethality screens in the context of WDR75 modulation

These integrated approaches can provide a systems-level understanding of WDR75's role in ribosome biogenesis and cellular stress responses.