wdr91 Antibody

What is WDR91 and what are its key cellular functions?

WDR91 (WD repeat domain 91) is a Rab7 effector protein that contains WD40 repeats which facilitate protein-protein interactions. Structurally, WDR91 consists of seven putative WD40 repeats at the C-terminus (aa 392-741) and a coiled-coil domain (aa 178-215) in the N-terminus . Functionally, WDR91 serves as a key factor that specifies the endosomal retrieval subdomain, which is essential for retromer-dependent recycling of endosomal cargoes . WDR91 acts as a negative regulator of PI3 kinase/PI3K activity associated with endosomal membranes via BECN1, regulating endosome fusion, recycling, sorting, and early-to-late endosome transport . Recent research also identifies WDR91 as a positive regulator of antisense oligonucleotide (ASO) activity, suggesting its importance in modulating therapeutic nucleic acid efficiency .

Where does WDR91 protein typically localize within cells?

Immunostaining studies reveal that only a small proportion of endogenous WDR91 colocalizes with the early endosome marker EEA1, while most WDR91 is colocalized with Rab7 or LAMP1 . In coexpression assays, GFP-WDR91 mainly colocalizes with late endosomal proteins Rab7, Rab9, and CD63, but not with early endosomal proteins EEA1 and Rab5 . WDR91 also localizes to a proportion of LAMP1-marked vesicles but does not localize to LysoTracker-positive lysosomes . This distinct localization pattern is crucial for researchers to consider when designing experiments involving WDR91 detection.

What molecular weight should I expect for WDR91 in Western blot analyses?

WDR91 antibodies typically detect proteins at two molecular weights:

Variations in observed molecular weight may occur depending on cell type, post-translational modifications, or degradation products. When troubleshooting WDR91 detection, researchers should consider running appropriate positive controls and blocking peptides to confirm specificity .

What factors should I consider when selecting a WDR91 antibody for my research?

When selecting a WDR91 antibody, researchers should consider:

• Target epitope region: Antibodies targeting different regions of WDR91 may yield different results. N-terminal antibodies may detect both full-length and truncated forms .

• Validated applications: Ensure the antibody has been validated for your specific application (WB, ICC/IF, IHC-P, IP, ELISA) .

• Species reactivity: Most available WDR91 antibodies react with human samples; verify cross-reactivity if working with other species .

• Immunogen used: Different antibodies use different immunogens (synthetic peptides or recombinant fragments), which may affect specificity .

• Validation data: Review published literature or manufacturer validation data showing specificity in relevant experimental contexts .

How can I validate the specificity of a WDR91 antibody?

To validate WDR91 antibody specificity:

• Knockout/knockdown controls: Use WDR91 knockout (KO-91) cells or WDR91 siRNA-treated cells as negative controls .

• Overexpression studies: Transfect cells with WDR91 expression constructs (e.g., Flag-WDR91, GFP-WDR91) and verify increased signal .

• Peptide competition: Pre-incubate the antibody with the immunizing peptide to confirm signal blocking.

• Cross-validation: Compare results using multiple antibodies targeting different epitopes of WDR91 .

• Immunoprecipitation followed by mass spectrometry: Confirm the identity of the immunoprecipitated protein .

What are the most reliable applications for WDR91 antibodies?

Based on the search results, WDR91 antibodies have been most extensively validated for:

How can I optimize Western blot protocols for WDR91 detection?

For optimal WDR91 Western blot detection:

• Sample preparation:

  • Use RIPA buffer with protease inhibitors for cell lysis

  • Load 15-50 μg of total protein per lane

• Gel electrophoresis and transfer:

  • Use 8-10% SDS-PAGE gels to effectively resolve the 83 kDa band

  • Transfer at 100V for 90 minutes to ensure complete transfer of higher molecular weight proteins

• Antibody incubation:

  • Primary antibody: 1:500-1:3000 dilution in 5% BSA-TBST, overnight at 4°C

  • Secondary antibody: HRP-conjugated anti-rabbit IgG at 1:5000-1:10000

• Detection:

  • Use ECL technique with 10-30 second exposure time for optimal results

  • For weaker signals, consider enhanced chemiluminescence substrates

What is the best approach for visualizing endogenous WDR91 in cellular compartments?

To effectively visualize WDR91 in cellular compartments:

• Co-immunostaining strategy:

  • Fix cells with 4% paraformaldehyde for 15 minutes

  • For colocalization studies, co-stain with markers for:

    • Late endosomes: Rab7, Rab9, CD63

    • Early endosomes: EEA1, Rab5

    • Lysosomes: LAMP1

• Live-cell imaging approaches:

  • Transfect cells with GFP-WDR91 or WDR91-GFP

  • Co-transfect with mCherry-tagged endosomal markers (e.g., 2×FYVE-mCh, mCh-CD63)

  • For studying active Rab7, use mCh-PH2C1 as a marker

• Analysis of endosomal subdomains:

  • Examine colocalization with SNX-retromer components (SNX27, VPS35)

  • Visualize F-actin organization using Phalloidin staining

  • Check ARPC2 and FAM21 distribution to assess WASH complex localization

How does WDR91 interact with Rab7 and what is the functional significance?

WDR91 interacts with Rab7 through its WD40 repeat domain located at the C-terminus:

• Interaction mechanism:

  • The WD40 repeats in WDR91 (aa 392-747) are necessary and sufficient for binding to Rab7

  • WDR91 is recruited to endosomes by active GTP-bound Rab7 (GTP-Rab7)

  • This interaction can be detected by co-immunoprecipitation using either WDR91 or Rab7 antibodies

• Functional significance:

  • WDR91 competes with VPS41 (a HOPS component) for binding to Rab7

  • This competition maintains appropriate levels of lysosome fusion

  • WDR91 promotes the interaction of Rab7 with SNX-retromer components, restricting Rab7 to endosomal retrieval subdomains

  • Loss of WDR91 causes abnormal enlargement of lysosomes by enhancing their fusion

What is the role of WDR91 in endosomal recycling pathways?

WDR91 plays a critical role in endosomal recycling through several mechanisms:

• Formation of endosomal retrieval subdomains:

  • WDR91 specifies the endosomal retrieval subdomain essential for retromer-dependent recycling

  • In WDR91-deficient cells, Rab7 and SNX-retromer components fail to localize to endosomal subdomains

• Regulation of cargo recycling:

  • WDR91 is required for recycling of cell surface receptors (e.g., β2AR) back to the plasma membrane

  • It facilitates endosome-to-Golgi recycling of intracellular receptors like CI-MPR

  • Loss of WDR91 causes trapping of SNX-retromer complexes on enlarged intermediate endosomes

• Actin organization on endosomes:

  • WDR91 interacts with FAM21 (a WASH complex component)

  • It promotes FAM21 enrichment at endosomal retrieval subdomains

  • In WDR91-deficient cells, F-actin evenly surrounds the enlarged endosomes, impairing endosomal actin organization

How do WDR91 and WDR81 cooperate in endosomal function?

WDR91 and WDR81 show functional relationship in endosomal pathways:

What phenotypes are observed in WDR91-deficient models and how can they be studied?

WDR91-deficient models display several phenotypes that can be studied using specific methods:

• Neuronal phenotypes:

  • Mice lacking Wdr91 in the central nervous system show progressive motor and behavioral defects

  • Marked neuronal loss occurs in cerebral and cerebellar cortices

  • Methods for analysis:

    • Behavioral testing (motor function, learning, memory)

    • Immunohistochemistry for activated Caspase 3 (AC3) to detect apoptotic cells

    • GFAP and Iba1 staining to assess astrocyte and microglial activation

• Cellular phenotypes:

  • Abnormal enlargement of lysosomes

  • Accumulation of autophagic cargoes and ubiquitin-positive inclusions

  • Methods for analysis:

    • Live-cell imaging of fluorescently labeled organelles

    • Electron microscopy to visualize ultrastructural changes

    • Treatment with vacuolin-1 to monitor lysosome enlargement kinetics

• Molecular phenotypes:

  • Defective early-to-late endosome conversion

  • Impaired endosomal recycling of membrane receptors

  • Methods for analysis:

    • Receptor recycling assays using β2AR or CI-MPR

    • Analysis of SNX-retromer localization by immunofluorescence

    • Examination of endosomal actin organization using Phalloidin staining

How is WDR91 implicated in antisense oligonucleotide (ASO) therapy effectiveness?

Recent research has identified WDR91 as a modulator of antisense oligonucleotide (ASO) activity:

• Discovery approach:

  • A genome-wide CRISPR knockout screen identified WDR91 as a positive regulator of ASO activity

  • The screen evaluated 19,114 coding genes using the Brunello CRISPR knockout library

  • WDR91 was one of the top candidates showing significant impact on ASO potency

• Validation methods:

  • siRNA-mediated knockdown of WDR91 significantly inhibited the anti-proliferative activity of tc-TSB-T3 (a tricyclo-ASO)

  • Cell viability was measured using crystal violet colorimetric assay 4 days after transfection

  • WDR91 showed the strongest effect among validated candidates (WBSCR16, ELMOD1, WDR91)

• Potential mechanisms and research implications:

  • WDR91's role in endosomal trafficking may influence ASO delivery to intended cellular compartments

  • Understanding WDR91 function could guide improvements in ASO design and delivery systems

  • This finding suggests targeting WDR91-related pathways could enhance ASO therapeutic efficiency

What are the most effective experimental approaches to study WDR91 protein-protein interactions?

To effectively study WDR91 protein-protein interactions:

• Co-immunoprecipitation (co-IP) approaches:

  • For endogenous interactions:

    • Immunoprecipitate with WDR91 antibody (3μg/mg lysate) from cell lysates

    • Detect interacting partners (Rab7, VPS35, SNX27, SNX6, SNX3, FAM21) by Western blot

  • For exogenous interactions:

    • Transfect cells with tagged constructs (GFP-WDR91, Flag-Rab7, etc.)

    • Immunoprecipitate using tag-specific antibodies and detect bound proteins

• Domain mapping strategies:

  • Generate truncation mutants of WDR91:

    • WDR91(1-405): N-terminal region containing coiled-coil domain

    • WDR91(392-747): C-terminal region containing WD40 repeats

  • Test interaction with specific partners to identify interaction domains

  • Point mutants like WDR91(3A) or WDR91(Δ5) can be used to disrupt specific interactions

• Functional interaction assays:

  • Competition assays between WDR91 and VPS41 for Rab7 binding

  • Rescue experiments in knockout cells:

    • Express wild-type or mutant WDR91 in KO-91 cells

    • Assess restoration of phenotypes like receptor recycling, SNX-retromer localization

  • Combined knockdown experiments to assess hierarchy in molecular pathways

What considerations are important when comparing results across different WDR91 knockout models?

When comparing results across different WDR91 knockout models:

• Model system differences:

  • Cell line models (HeLa KO-91) vs. animal models (Wdr91 cKO mice)

  • Tissue-specific knockout (e.g., CNS-specific) vs. whole-organism knockout

  • Acute (siRNA) vs. chronic (genetic) knockout effects

• Knockout verification approaches:

  • Western blot confirmation using validated WDR91 antibodies

  • Genomic verification of CRISPR-mediated alterations

  • Transcript analysis by RT-qPCR

• Phenotype assessment standardization:

  • Endosomal morphology: Measure size and distribution of endosomes using consistent markers

  • Receptor recycling: Standardize agonist concentrations and time points for β2AR recycling assays

  • Lysosomal function: Use standardized assays for pH, enzyme activity, or degradative capacity

• Compensatory mechanisms:

  • Assess WDR81 expression in WDR91 knockout models

  • Consider potential upregulation of alternative Rab7 effectors

  • Evaluate possible adaptation in long-term knockout models vs. acute depletion

How can I resolve common issues with WDR91 detection in Western blot?

Common issues with WDR91 detection and their solutions:

What are the key considerations for studying WDR91 in primary neurons?

For studying WDR91 in primary neurons:

• Expression and detection:

  • WDR91 is highly expressed in neurons and essential for neuronal development

  • For immunofluorescence, use 4% paraformaldehyde fixation followed by 0.1% Triton X-100 permeabilization

  • Co-stain with neuronal markers (MAP2, NeuN) and endosomal/lysosomal markers

• Knockout/knockdown approaches:

  • For acute depletion, use siRNA or shRNA with validated targeting sequences

  • For genetic models, consider Cre-loxP systems for conditional neuronal knockout (e.g., Nestin-Cre)

  • Assess phenotypes: enlarged lysosomes, accumulation of autophagic cargoes, axonal swelling

• Functional assays:

  • Monitor lysosomal volume and function using LysoTracker or LysoSensor

  • Assess autophagy flux using LC3 and p62 markers

  • Examine receptor trafficking using neuron-specific receptors (e.g., TrkB, AMPA receptors)

• Neuronal viability considerations:

  • Monitor activated Caspase 3 to detect apoptotic neurons

  • Assess neurite growth and branching as indicators of neuronal health

  • Consider longer-term consequences of WDR91 deficiency on neuronal survival