WDR73 Antibody

What is WDR73 and why is it significant for research?

WDR73 is a WD40-repeat-containing protein encoded by the WDR73 gene (also known by aliases GAMOS, HSPC264). This protein is of particular interest because loss-of-function mutations in WDR73 have been identified as causative for Galloway-Mowat syndrome, a rare autosomal-recessive condition characterized by the co-occurrence of neurological defects and glomerular-renal disease . The significance of WDR73 extends beyond its association with this syndrome, as recent research has implicated it in several crucial cellular pathways including the processing of uridylate-rich small nuclear RNAs (UsnRNA), transcriptional response regulation, and cell cycle control . WDR73 is present in both brain and kidney tissues and localizes diffusely in the cytoplasm during interphase but relocates to spindle poles and astral microtubules during mitosis .

What are the key characteristics of commercially available WDR73 antibodies?

Commercial WDR73 antibodies, such as the polyclonal antibody from Thermo Fisher (PA5-58751), are typically raised against specific immunogen sequences. For example, the aforementioned antibody targets the immunogen sequence: "ISGFDGTVQV YDATSWDGTR SQDGTRSQVE PLFTHRGHIF LDGNGMDPAP LVTTHTWHPC RPRTLLSATN DASLHVWDW" . When selecting an antibody, researchers should consider species reactivity (some show cross-reactivity with mouse and rat orthologs, with sequence identity around 73% and 71% respectively) . Most research-grade antibodies are specifically designated "For Research Use Only" and not for diagnostic procedures or resale without authorization . Before beginning experiments, researchers should verify the UniProt ID (Human: Q6P4I2) and Entrez Gene ID (Human: 84942) to ensure targeting the correct protein .

How should I validate a WDR73 antibody for my research application?

Validation of WDR73 antibodies requires a multi-step approach to ensure specificity and reliability in your experimental system:

• Western Blot Validation: Run western blots with positive controls (tissues known to express WDR73, like kidney or brain tissue) alongside negative controls. The WDR73 protein should appear at the expected molecular weight.

• Knockout/Knockdown Controls: Compare antibody reactivity in wild-type cells versus WDR73 knockout or knockdown models. Several studies have successfully created WDR73 knockout HEK293 cells using CRISPR/Cas9 targeting the region encompassing exon 6 .

• Multiple Antibody Validation: Use multiple antibodies targeting different epitopes of WDR73 to confirm specificity.

• Immunoprecipitation Analysis: Perform immunoprecipitation followed by mass spectrometry to confirm the antibody is capturing the intended protein.

• Immunocytochemistry Correlation: For cellular localization studies, verify that the staining pattern aligns with known WDR73 localization (cytoplasmic during interphase, spindle poles during mitosis) .

Several published studies have used anti-WDR73 antibodies from manufacturers such as Sigma-Aldrich for immunoblotting and immunofluorescence applications .

What are the optimal protocols for using WDR73 antibodies in immunofluorescence studies?

For optimal immunofluorescence studies with WDR73 antibodies, consider the following methodological approach:

• Cell Preparation:

  • For adherent cells like fibroblasts or podocytes, culture on Lab-Tek Chamber Slides or coverslips coated with rat-tail collagen type I .

  • Allow 48 hours of culture for appropriate cell attachment and protein expression.

• Fixation Options:

  • Cold 100% methanol fixation is recommended for preserving cytoskeletal elements and studying WDR73's interaction with microtubules.

  • Alternatively, use 4% paraformaldehyde (PFA) fixation with subsequent NH₄Cl (50 mM) treatment to quench free aldehyde groups .

• Blocking and Antibody Incubation:

  • Block with 10% normal serum in PBS to prevent non-specific binding.

  • Incubate with the anti-WDR73 primary antibody (1:100-1:500 dilution range) overnight at 4°C.

  • For co-localization studies with tubulin or other cytoskeletal elements, include antibodies such as anti-α-tubulin (Sigma-Aldrich) or anti-γ-tubulin (Santa Cruz) .

• Detection System:

  • For brightfield microscopy, use an avidin-biotin peroxidase kit with DAB as chromogen.

  • For fluorescence, use appropriate fluorophore-conjugated secondary antibodies.

  • Counterstain nuclei with DAPI or Hoechst.

• Specialized Applications:

  • For mitotic spindle localization studies, synchronize cells using nocodazole release protocol prior to fixation.

  • When studying focal adhesion, co-stain with adhesion markers and analyze using confocal microscopy.

How can I use WDR73 antibodies to investigate protein interactions with the Integrator complex?

To investigate WDR73 interactions with the Integrator complex components (particularly INTS9 and INTS11) , consider these approaches:

• Co-immunoprecipitation (Co-IP):

  • Prepare cell lysates using a lysis buffer containing 50 mM Tris-HCl, 150 mM NaCl, 0.5% sodium deoxycholate, 2 mM EDTA, 1% Triton X-100, and 0.1% SDS .

  • Immunoprecipitate with anti-WDR73 antibodies and probe for Integrator components, or vice versa.

  • Include appropriate negative controls (IgG or irrelevant antibodies).

  • For increased specificity, use tagged versions of WDR73 (e.g., 3×HA-WDR73) expressed in relevant cell lines .

• Proximity Ligation Assay (PLA):

  • This technique allows visualization of protein-protein interactions in situ.

  • Use antibodies against WDR73 and Integrator components from different species.

  • Apply species-specific secondary antibodies with attached DNA oligonucleotides.

  • Interaction proximity (< 40 nm) enables ligation and amplification, visualized as fluorescent spots.

• GST Pulldown Assays:

  • Express recombinant WDR73 as a His-tagged fusion protein and potential interacting partners as GST-fusion proteins .

  • Perform pulldown experiments and analyze by immunoblotting.

  • This approach has been successfully used to validate WDR73 interactions with other proteins like PIP4K2C .

• Mass Spectrometry Analysis:

  • After immunoprecipitation with WDR73 antibodies, perform mass spectrometry to identify associated proteins.

  • Compare results with control immunoprecipitations to identify specific interactors.

  • Validate findings with targeted Co-IP or GST pulldown assays.

What approaches can I use to investigate WDR73's role in cell cycle regulation?

WDR73 suppression leads to altered expression of genes encoding cell cycle regulatory proteins and G2/M phase arrest . To investigate this role:

• Cell Cycle Analysis with Flow Cytometry:

  • Generate WDR73 knockout or knockdown cell lines (e.g., using CRISPR/Cas9 or shRNA) .

  • Fix cells, stain with propidium iodide or DAPI, and analyze DNA content by flow cytometry.

  • Compare cell cycle distribution between WDR73-depleted and control cells.

• Immunofluorescence Analysis of Mitotic Cells:

  • Use WDR73 antibodies alongside markers for mitotic phases (phospho-histone H3, cyclin B).

  • Assess microtubule organization using α-tubulin antibodies.

  • Analyze spindle pole localization using γ-tubulin antibodies .

• Live Cell Imaging:

  • Express fluorescently-tagged WDR73 in cells and perform time-lapse imaging.

  • Track WDR73 localization during cell cycle progression.

  • Quantify mitotic duration and abnormalities in WDR73-depleted cells.

• Gene Expression Analysis:

  • Perform RNA-seq or qPCR to identify cell cycle genes affected by WDR73 depletion.

  • Validate protein-level changes by western blotting.

  • Correlate expression changes with cell cycle phenotypes.

• Rescue Experiments:

  • Reintroduce wild-type WDR73 into knockout cells to confirm specificity of cell cycle defects.

  • Use constructs carrying WT WDR73 and select stable integrants using hygromycin selection .

How can I address specificity issues when using WDR73 antibodies in different experimental contexts?

When addressing specificity issues with WDR73 antibodies:

• Antibody Validation in Your System:

  • Always validate commercial antibodies in your specific experimental system.

  • For HEK293 cells, CRISPR/Cas9 knockout models have been successfully generated and can serve as negative controls .

  • For tissue sections, compare staining patterns with in situ hybridization data.

• Cross-Reactivity Considerations:

  • Be aware of species differences - human WDR73 shares approximately 73% sequence identity with mouse orthologs and 71% with rat orthologs .

  • When using antibodies across species, validate with appropriate negative controls.

  • Consider using monoclonal antibodies for higher specificity in cross-species applications.

• Alternative Approaches:

  • For detection in cells or tissues where antibody specificity is challenging, consider tagged overexpression systems.

  • Epitope tags like HA or FLAG can be used with commercially validated tag antibodies .

  • For interaction studies, protein microarray approaches using purified recombinant His-WDR73 fusion proteins have been successful .

• Protocol Optimization:

  • Adjust blocking conditions (duration, blocking agent) to reduce non-specific binding.

  • Titrate antibody concentration to find optimal signal-to-noise ratio.

  • Consider antigen retrieval methods for tissue sections, as WDR73 epitopes may be masked.

What are the best practices for analyzing WDR73's role in focal adhesion pathways?

WDR73 depletion has been linked to impaired cell adhesion and focal adhesion pathways . To effectively study this relationship:

• Combined Transcriptomic and Proteomic Approach:

  • Generate WDR73 knockout cell lines (e.g., in HEK293 cells) using CRISPR/Cas9 .

  • Perform RNA-seq to identify differentially expressed genes enriched in focal adhesion pathways.

  • Complement with proteomic analysis to identify changes at the protein level.

• Morphological Analysis:

  • Quantify cell adhesion parameters in WDR73-depleted cells vs. controls.

  • Document and measure pseudopodia formation and cell spreading.

  • Use phase contrast and immunofluorescence imaging for detailed morphological assessment .

• Focal Adhesion Visualization:

  • Immunostain for focal adhesion markers (paxillin, vinculin, FAK).

  • Perform quantitative analysis of focal adhesion size, number, and distribution.

  • Use super-resolution microscopy for detailed structural analysis.

• Functional Assays:

  • Conduct cell adhesion assays on different substrates.

  • Perform cell migration assays (wound healing, transwell) to assess functional consequences.

  • Analyze cytoskeletal dynamics using live cell imaging.

• Interaction Analysis with PIP4K2C:

  • Investigate the WDR73-PIP4K2C interaction, as this has been validated to play a role in focal adhesion pathways .

  • Use co-immunoprecipitation with WDR73 antibodies to pull down PIP4K2C.

  • Perform GST pulldown assays using recombinant proteins to confirm direct interaction .

How can WDR73 antibodies contribute to understanding Galloway-Mowat syndrome pathogenesis?

Galloway-Mowat syndrome (GAMOS) is caused by loss-of-function mutations in WDR73 . WDR73 antibodies can help elucidate disease mechanisms through:

• Comparative Tissue Studies:

  • Analyze WDR73 expression and localization in normal versus patient-derived tissues.

  • Use immunohistochemistry with anti-WDR73 antibodies on kidney and brain sections.

  • Compare with other markers to understand tissue-specific pathologies.

• Patient-Derived Cell Models:

  • Study fibroblasts from affected individuals (as in the A-II-4 fibroblasts described in the literature) .

  • Analyze nuclear morphology, cell viability, and microtubule network organization.

  • Compare protein expression profiles using WDR73 and related pathway antibodies.

• Mechanistic Studies in Disease Models:

  • Use Wdr73 conditional knockout mouse models to study tissue-specific effects .

  • Apply WDR73 antibodies to track protein expression and localization in podocytes and neurons.

  • Correlate molecular findings with histopathological and functional outcomes.

• Therapeutic Target Identification:

  • Identify proteins that interact with WDR73 using immunoprecipitation followed by mass spectrometry.

  • Investigate compensatory mechanisms in WDR73-deficient cells.

  • Screen for compounds that might restore function in cells with WDR73 mutations.

What emerging techniques might enhance the study of WDR73's role in RNA processing?

WDR73 interacts with the Integrator complex and influences UsnRNA processing . To advance this research:

• CLIP-seq (Cross-linking Immunoprecipitation with Sequencing):

  • Use WDR73 antibodies to immunoprecipitate RNA-protein complexes after UV crosslinking.

  • Sequence bound RNAs to identify direct WDR73 RNA targets.

  • Compare results between normal and disease contexts.

• RNA-protein Interaction Mapping:

  • Apply techniques like RNA Antisense Purification (RAP) to identify proteins associated with specific UsnRNAs.

  • Use WDR73 antibodies to detect its presence in these complexes.

  • Map domains of interaction between WDR73 and RNA processing machinery.

• Live Cell RNA Imaging:

  • Combine MS2-tagged UsnRNAs with fluorescently labeled WDR73 to visualize dynamics in living cells.

  • Track processing events in real-time using advanced microscopy.

  • Compare processing kinetics in WDR73-normal versus depleted conditions.

• Integrator Complex Analysis:

  • Investigate how WDR73 affects assembly and function of the Integrator complex.

  • Use antibodies against WDR73, INTS9, and INTS11 to study complex formation .

  • Apply structural biology approaches to understand molecular interactions.