WDR92 Antibody

What is WDR92 and what are its primary functions in cellular biology?

WDR92, also known as monad or DNAAF10 (Dynein axonemal assembly factor 10), is a WD40 repeat-containing protein that functions primarily in axonemal dynein assembly. It contains seven WD repeats forming a β-propeller structure that facilitates protein-protein interactions . WDR92 is essential for the cytoplasmic stability of axonemal dynein heavy chains, playing a crucial role in cilia formation and function . Additionally, studies have shown that WDR92, together with its binding partner RNA polymerase II-associated protein 3 (RPAP3), may function as a modulator of apoptosis, as overexpression of these proteins potentiates apoptosis and caspase-3 activation induced by TNF-α and cycloheximide .

Research methodologies to investigate WDR92 function typically include gene knockdown/knockout studies, protein interaction assays, and ciliary function assessments. The Chlamydomonas model has proven particularly valuable for understanding WDR92's role in dynein assembly and ciliary function .

What is the tissue distribution of WDR92 expression?

WDR92 is widely expressed in human tissues, with particularly high expression in testis . When studying tissue expression patterns, researchers should consider using multi-tissue Western blots with appropriate controls. The following table summarizes tissues where WDR92 antibodies have been validated for detection:

For optimal detection in tissue samples, antigen retrieval with TE buffer pH 9.0 is suggested, although citrate buffer pH 6.0 may be used as an alternative for certain applications .

What applications are most suitable for WDR92 antibody detection?

WDR92 antibodies have been validated for multiple applications, with specific dilution recommendations for each technique. The most suitable applications include:

When designing experiments, it is critical to optimize antibody dilutions for each specific system to obtain optimal results. Sample-dependent variations may require further titration beyond the recommended ranges .

How should I optimize Western blot conditions for detecting WDR92?

For optimal Western blot detection of WDR92:

• Prepare samples in standard SDS-PAGE loading buffer with reducing agent

• Use 10-12% polyacrylamide gels for good resolution around the 40 kDa range

• Transfer to PVDF or nitrocellulose membranes using standard protocols

• Block with 5% non-fat milk or BSA in TBST

• Apply primary WDR92 antibody at 1:500-1:1000 dilution

• Incubate overnight at 4°C for best sensitivity

• Use appropriate HRP-conjugated secondary antibody

• Develop using enhanced chemiluminescence

When interpreting results, the expected molecular weight for WDR92 is 40 kDa, consistent with the calculated molecular weight from its 357-358 amino acid sequence . K-562 cells can serve as a positive control for Western blot applications .

What are the recommended sample preparation methods for immunohistochemistry?

For optimal WDR92 detection in tissue sections:

• Fix tissues in 10% neutral buffered formalin

• Process and embed in paraffin following standard protocols

• Section tissues at 4-6 μm thickness

• Perform antigen retrieval using TE buffer pH 9.0 (preferred) or citrate buffer pH 6.0

• Block endogenous peroxidase activity and non-specific binding

• Apply WDR92 antibody at 1:50-1:1000 dilution

• Incubate overnight at 4°C

• Apply appropriate detection system

• Counterstain, dehydrate, and mount

Human colon tissue, human testis tissue, and mouse testis tissue have been validated as positive controls for IHC applications . When interpreting results, consider that WDR92 may show both cytoplasmic and nuclear localization, depending on the cell type.

How does WDR92 contribute to ciliary assembly and function?

WDR92 plays a critical role in ciliary assembly through its function in axonemal dynein heavy chain stability. Studies in Chlamydomonas have shown that:

• WDR92 mutants build only ~0.7-μm cilia lacking both inner and outer dynein arms

• The mutant cilia maintain intact doublet microtubules and central pair structures

• WDR92 is specifically required for the stability of axonemal dynein heavy chains in the cytoplasm

• WDR92 mutants show severely reduced amounts of all three outer arm heavy chains

These findings suggest WDR92 acts at a specific step in the dynein assembly pathway, distinct from the folding pathway used by cytoplasmic/IFT dynein heavy chains . Research methodologies for studying WDR92's role in ciliary function include:

• Ciliary beat frequency measurements

• Transmission electron microscopy of ciliary cross-sections

• Immunofluorescence to visualize ciliary components

• Biochemical fractionation of cytoplasmic dynein assembly intermediates

What protein complexes does WDR92 participate in?

WDR92 associates with several protein complexes involved in dynein assembly:

• A prefoldin-like cochaperone complex

• The R2TP complex

• Known dynein assembly factors

Research in Chlamydomonas has revealed that:

• In control cytoplasm, outer arm dynein components are present in several distinct high molecular weight complexes

• In wdr92-1 mutants, all three outer arm heavy chains are almost completely absent

• The IFT dynein heavy chain remains present in normal amounts

• The DYX1C1/PF23 (DNAAF4) assembly factor shows altered oligomeric status in wdr92 mutants

• In the absence of WDR92, the high molecular weight form of DYX1C1 is completely absent

These findings suggest WDR92 is part of a specific pathway for axonemal dynein assembly . To study WDR92-containing complexes, researchers typically employ:

• Co-immunoprecipitation

• Gel filtration chromatography

• Mass spectrometry analysis

• Yeast two-hybrid screening

How does WDR92 interact with RPAP3 in apoptotic pathways?

WDR92 has been found to interact with RNA polymerase II-associated protein 3 (RPAP3), with implications for apoptotic regulation. Overexpression studies have shown that:

• Increased expression of either WDR92 or RPAP3 potentiates apoptosis

• This overexpression enhances caspase-3 activation induced by TNF-α and cycloheximide

• WDR92 may function together with RPAP3 as a novel modulator of apoptosis

To investigate this interaction, researchers can employ:

• Co-immunoprecipitation to confirm direct interaction

• RNAi-mediated knockdown to assess functional relationships

• Apoptosis assays measuring caspase activation

• Localization studies using fluorescently tagged proteins

• Flow cytometry to quantify apoptotic populations

The specific mechanisms linking WDR92's role in ciliary assembly with its potential function in apoptosis remain an area for further investigation .

What are the recommended fixation and permeabilization methods for WDR92 immunofluorescence?

For optimal immunofluorescence detection of WDR92:

• Culture cells on coverslips or chamber slides to 70-80% confluence

• Fix cells using one of the following methods:

  • 4% paraformaldehyde (10-15 minutes at room temperature)

  • Methanol (10 minutes at -20°C) for membrane permeabilization

• If using paraformaldehyde, permeabilize with 0.1-0.5% Triton X-100 for 5-10 minutes

• Block with 1-5% BSA or normal serum in PBS for 30-60 minutes

• Apply WDR92 antibody at a 1:10-1:100 dilution

• Incubate overnight at 4°C or 1-2 hours at room temperature

• Apply fluorophore-conjugated secondary antibody

• Counterstain nuclei with DAPI

• Mount with anti-fade mounting medium

HepG2 cells have been validated as a positive control for IF/ICC applications . When imaging, consider that WDR92 may show both cytoplasmic and nuclear localization patterns.

How can I validate the specificity of a WDR92 antibody?

To validate the specificity of a WDR92 antibody:

• Positive controls: Use known WDR92-expressing tissues/cells:

  • K-562 cells for Western blot

  • Human testis tissue for IHC

  • HepG2 cells for IF/ICC

• Molecular weight verification: Confirm detection at the expected 40 kDa size on Western blots

• Knockdown/knockout validation: Compare antibody signal in:

  • WDR92 siRNA-treated cells

  • CRISPR/Cas9 WDR92-knockout cells

  • Genetic models (e.g., Chlamydomonas wdr92-1 mutant)

• Peptide competition: Pre-incubate antibody with immunizing peptide to block specific binding

• Cross-species reactivity: Test against samples from different species:

  • Human (58% identical to Chlamydomonas WDR92)

  • Mouse (93% antigen sequence identity to human)

  • Rat (92% antigen sequence identity to human)

What are the optimal storage conditions for WDR92 antibodies?

For maximum stability and longevity of WDR92 antibodies:

• Store at -20°C according to manufacturer recommendations

• Antibodies in PBS with 0.02% sodium azide and 50% glycerol (pH 7.3) are stable for one year after shipment

• Aliquoting is generally unnecessary for -20°C storage

• Avoid repeated freeze-thaw cycles

• Some antibody preparations (e.g., 20μl sizes) may contain 0.1% BSA for additional stability

• For working solutions, store at 4°C for up to one month

• Check specific product documentation for any variations in storage recommendations

How should differences in WDR92 expression across tissues and experimental conditions be interpreted?

When analyzing WDR92 expression:

• Baseline expression: Consider that WDR92 is widely expressed, with particularly high levels in testis

• Functional context: Interpret expression in relation to:

  • Ciliated tissue requirements

  • Dynein assembly needs

  • Cell cycle stage

  • Apoptotic conditions

• Quantification approaches:

  • For Western blots: Normalize to loading controls (β-actin, GAPDH)

  • For IHC: Use scoring systems based on staining intensity and percentage of positive cells

  • For IF: Measure mean fluorescence intensity and subcellular distribution

• Comparative analysis:

  • Between normal and pathological tissues

  • Across developmental stages

  • In response to experimental manipulations

• Validation with multiple techniques: Confirm protein-level changes with mRNA expression data when possible

What are the critical parameters for analyzing WDR92 localization patterns?

When analyzing subcellular localization of WDR92:

• Expected patterns:

  • Cytoplasmic distribution consistent with its role in dynein assembly

  • Potential nuclear localization in specific cell types

  • Association with ciliary basal bodies in ciliated cells

• Co-localization studies: Consider dual staining with:

  • Basal body markers (γ-tubulin)

  • Ciliary markers (acetylated tubulin)

  • Other dynein assembly factors (DYX1C1/DNAAF4)

  • RPAP3 (binding partner in apoptotic pathways)

• Quantification approaches:

  • Pearson's correlation coefficient for co-localization analysis

  • Line scan intensity profiles across cellular structures

  • Quantification of nuclear vs. cytoplasmic signal intensity

• Technical considerations:

  • Optical sectioning (confocal microscopy) for precise localization

  • Super-resolution techniques for detailed structural associations

  • Live cell imaging for dynamic localization studies

• Functional correlation: Connect localization patterns with:

  • Ciliary assembly status

  • Cell cycle stage

  • Apoptotic conditions

When interpreting localization data, consider that alterations in WDR92 distribution may indicate dysfunction in dynein assembly pathways or changes in its association with protein complexes .

What are potential research applications of WDR92 antibodies in studying ciliopathies?

WDR92 antibodies offer valuable tools for investigating ciliopathies—disorders resulting from ciliary dysfunction:

• Diagnostic potential:

  • Assess WDR92 expression and localization in patient samples

  • Screen for alterations in dynein assembly pathways

• Mechanistic studies:

  • Investigate WDR92's role in primary ciliary dyskinesia

  • Examine relationships between WDR92 mutations and ciliary phenotypes

  • Characterize WDR92-containing protein complexes in disease states

• Therapeutic development:

  • Identify compounds that modulate WDR92 function

  • Screen for molecules that stabilize dynein assembly in WDR92-deficient cells

• Model systems approaches:

  • Generate tissue-specific WDR92 knockout models

  • Create knock-in models of patient-derived WDR92 mutations

  • Develop human iPSC-derived organoid models to study WDR92 in ciliated tissues

Research methodologies should include comprehensive phenotypic analysis of ciliary structure and function, combined with molecular characterization of WDR92-dependent assembly pathways .

How might WDR92's dual role in ciliary assembly and apoptosis be mechanistically linked?

The dual role of WDR92 in both ciliary assembly and potential apoptotic regulation presents intriguing research opportunities:

• Hypothesis generation:

  • WDR92 may function as a cellular sensor linking ciliary integrity with cell survival

  • Disruption of protein homeostasis might trigger both ciliary defects and apoptotic signaling

  • The R2TP complex association may represent a common pathway affecting both processes

• Experimental approaches:

  • Identify shared interaction partners between ciliary and apoptotic pathways

  • Characterize WDR92 isoforms or post-translational modifications specific to each function

  • Develop conditional expression systems to temporally separate WDR92's functions

• Technical considerations:

  • Time-course studies to determine sequence of events

  • Domain mapping to identify regions critical for specific functions

  • Proximity labeling techniques to capture context-specific interaction networks