WDR35 Antibody

What is WDR35 and what cellular functions does it perform?

WDR35 (WD Repeat Domain 35) is an essential component of cilia with critical functions in ciliogenesis and intraflagellar transport. Research has demonstrated that WDR35 localizes primarily to the periciliary region in cells, with partial colocalization with γ-tubulin . Additionally, WDR35 is detected along cilia axonemes, where it colocalizes with acetylated α-tubulin .

The protein is highly enriched in ciliated tissues, particularly in the developing lung and nervous system during embryonic development . Functionally, WDR35 serves as a component of the IFT-A (Intraflagellar Transport A) complex and is crucial for its stability . Recent evidence indicates that WDR35, likely with other IFT proteins, acts as a COPI-like complex to deliver proteins to growing cilia . Knockout or mutation of WDR35 results in severe ciliogenesis defects, underscoring its essential role in ciliary assembly and function .

What epitopes are typically targeted in WDR35 antibodies?

Commercial WDR35 antibodies are typically designed to target several distinct epitope regions of the protein:

• N-terminal region antibodies:

  • AA 60-110 (N-Term)

  • AA 71-120

• Mid-region antibodies:

  • Currently less represented in available antibodies

• C-terminal region antibodies:

  • AA 871-1170

  • AA 954-1181

When selecting a WDR35 antibody, researchers should consider which domain of the protein is most relevant to their research question. The N-terminal β-propeller domain of WDR35 is involved in higher complex organization of transport modules , while other regions may have different structural or functional significance.

What species reactivity can be expected from WDR35 antibodies?

WDR35 antibodies show varied cross-reactivity across species due to the high conservation of the protein sequence. Based on sequence homology analysis:

When selecting a WDR35 antibody for non-human studies, researchers should verify the predicted reactivity based on BLAST analysis of the target epitope sequence . For example, antibody ABIN6744204 (targeting AA 71-120) shows high predicted reactivity across mammals and even zebrafish models, making it suitable for comparative studies .

How can WDR35 antibodies be used to visualize ciliary structures?

WDR35 antibodies are valuable tools for visualizing ciliary structures across multiple experimental systems:

Cell culture applications:

• Localization studies: WDR35 antibodies can be used to detect the protein at centrosomes and basal bodies, as well as along the ciliary axoneme in cultured cells such as NIH 3T3 and IMCD3 .

• Co-localization studies: Combine WDR35 antibodies with other ciliary markers such as:

  • γ-tubulin for basal bodies/centrosomes

  • Acetylated α-tubulin for ciliary axonemes

Tissue section applications:
WDR35 antibodies can label both primary and specialized cilia in tissue sections, with particularly strong staining in highly ciliated tissues such as developing lung and nervous system . For optimal results in tissue immunohistochemistry:

• Use thin sections (0.4 μm) for confocal imaging

• Employ nuclear counterstains (e.g., TOTO-3) for structural context

• Include additional ciliary markers for co-localization analyses

What methodological approaches can verify WDR35 antibody specificity?

Verifying antibody specificity is critical for reliable experimental results. For WDR35 antibodies, multiple validation methods should be employed:

• Genetic knockout/mutant controls:

  • WDR35 antibody staining should be absent or significantly reduced in WDR35 mutant/knockout samples

  • This has been demonstrated in both yeti mutant mouse embryo sections and WDR35Δ5 patient fibroblasts

• siRNA knockdown validation:

  • Reduction of WDR35 staining should correlate with siRNA-mediated reduction of Wdr35 mRNA

  • Studies have shown ~85% reduction in Wdr35 mRNA resulting in correspondingly reduced antibody staining

• Peptide competition assays:

  • Pre-adsorption of the antibody with the immunizing peptide should abolish specific staining

  • This approach has been documented for WDR35 antibodies to confirm specificity

• Multiple antibody validation:

  • Use of independent antibodies targeting different epitopes of WDR35 should show consistent localization patterns

  • Research has employed two independent antibodies directed against different epitopes with consistent results

How can WDR35 antibodies be used to study ciliopathies?

WDR35 antibodies are valuable tools for studying ciliopathies, particularly short-rib polydactyly (SRP) syndromes and Sensenbrenner syndrome/Cranioectodermal dysplasia (CED):

• Diagnostic applications:

  • WDR35 antibodies can help assess WDR35 protein expression levels and localization in patient-derived cells

  • In WDR35Δ5 SRP fibroblasts, only remnant expression of endogenous WDR35Δ5 was detected, confirming the loss-of-function nature of the mutation

• Functional studies:

  • Combine WDR35 antibodies with other ciliary markers to assess cilia formation defects

  • Researchers can quantify ciliation rates and cilia morphology in control versus patient cells

• Mechanistic investigations:

  • Use WDR35 antibodies to examine the consequences of disease-associated mutations on:

    • Protein stability and expression

    • Subcellular localization

    • Interaction with other IFT-A components

How can WDR35 antibodies help investigate the IFT-A complex?

WDR35 antibodies enable sophisticated analyses of IFT-A complex composition and function:

• Co-immunoprecipitation studies:

  • WDR35 antibodies can be used to pull down the entire IFT-A complex

  • This approach can reveal which components associate with WDR35 and how mutations affect complex assembly

• Comparative analysis of complex stability:

  • Research has revealed that in Wdr35-/- embryos, the IFT-A complex is destabilized

  • Both non-core components (IFT139 and IFT43) are missing and core components (IFT122 and IFT144) are significantly reduced in the purified mutant complex

• Differential protein expression analysis:

  • WDR35 antibodies can be used in immunoblotting to compare total cellular levels of IFT-A components between wildtype and mutant samples

  • This has revealed that WDR35 is required for stability of its non-core components

What methodological approaches are used to study WDR35's role in vesicle transport?

Advanced imaging and biochemical techniques using WDR35 antibodies have revealed its role in vesicular transport to cilia:

• Correlative light and electron microscopy (CLEM):

  • WDR35-EmGFP expression combined with TEM has demonstrated WDR35's association with electron-dense vesicles at the ciliary base

  • This approach revealed that WDR35 is required for the formation of coated vesicles destined to deliver membrane cargos to cilia

• Immunogold labeling for electron microscopy:

  • WDR35 antibodies conjugated to gold particles can localize the protein at ultrastructural resolution

  • This technique has revealed WDR35-positive particles at the cilia base, within the axoneme, and around vesicles at the ciliary sheath

• Rescue experiments with tagged WDR35:

  • Expression of WDR35-EmGFP in Wdr35-/- MEFs rescues cilia phenotypes and restores the presence of electron-dense vesicles at the ciliary base

  • This methodological approach confirms the specific requirement for WDR35 in vesicle coating and transport

How can WDR35 antibodies be used to study protein trafficking to cilia?

WDR35 antibodies can be employed in multiple experimental approaches to investigate protein trafficking to cilia:

• Co-localization with cargo proteins:

  • WDR35 antibodies can be used alongside antibodies against putative ciliary cargo proteins to assess their co-trafficking

  • This approach can help identify which proteins depend on WDR35 for ciliary localization

• Live-cell imaging combined with immunofluorescence:

  • Live imaging of fluorescently-tagged cargo proteins followed by fixation and WDR35 immunostaining

  • This allows correlation between dynamic trafficking events and WDR35 localization

• Proximity labeling approaches:

  • WDR35 antibodies can be used to validate results from proximity labeling experiments (BioID, APEX) identifying proteins in close proximity to WDR35 during trafficking

What are common challenges when using WDR35 antibodies in immunofluorescence?

Researchers may encounter several challenges when using WDR35 antibodies for immunofluorescence:

• Variable signal intensity along cilia:

  • WDR35 typically shows intense staining at the ciliary base with fainter punctate staining along the axoneme

  • Solution: Use confocal microscopy with appropriate exposure settings to capture both intense and faint signals

• Background staining:

  • Some WDR35 antibodies may show nonspecific cytoplasmic staining

  • Solution: Include proper controls (peptide competition, WDR35 mutant/knockdown samples) to distinguish specific from nonspecific signals

• Detection in highly ciliated tissues:

  • While enriched in ciliated tissues, individual cilia may still show relatively weak WDR35 staining

  • Solution: Use thin sections (0.4 μm) for tissue immunohistochemistry and employ signal amplification methods if needed

How should researchers select the most appropriate WDR35 antibody for their specific application?

When selecting a WDR35 antibody, researchers should consider:

• Epitope location:

  • For studying N-terminal domain function or mutations, choose antibodies targeting AA 60-120

  • For C-terminal studies, select antibodies targeting AA 871-1181

  • Use antibodies targeting different regions to confirm results

• Application compatibility:

  • Verify the antibody is validated for your specific application (WB, IF, IHC, IP)

  • For example, antibody ABIN6744204 is validated for Western blotting

• Species compatibility:

  • Confirm the antibody recognizes WDR35 in your experimental model

  • Many antibodies show cross-reactivity (e.g., 92% identity between human and mouse WDR35)

• Mutant compatibility:

  • For studies of specific WDR35 mutations, ensure the antibody's epitope is not affected by the mutation

  • For example, antibodies targeting regions outside exon 5 would still recognize the WDR35Δ5 mutant protein

What controls are essential when using WDR35 antibodies in experimental studies?

To ensure reliable results with WDR35 antibodies, researchers should include these controls:

• Negative controls:

  • WDR35 knockout/mutant samples (when available)

  • siRNA knockdown samples

  • Peptide competition/pre-adsorption

  • Secondary antibody-only controls

• Positive controls:

  • Known WDR35-expressing cells (e.g., ciliated IMCD3 cells)

  • Tissues with high ciliary density (developing lung, brain)

  • Rescue experiments (e.g., WDR35-GFP expression in mutant cells)

• Co-localization controls:

  • Co-staining with established ciliary markers:

    • γ-tubulin for basal bodies

    • Acetylated α-tubulin for axonemes

    • Other IFT-A components for complex formation