Phospho-WASF1 (Tyr125) Antibody

What is WASF1 and what cellular functions does it regulate?

WASF1 (also known as WAVE1) is a member of the Wiskott-Aldrich syndrome protein (WASP) family that functions as a downstream effector molecule in signal transduction pathways. It plays a critical role in regulating the actin cytoskeleton by mediating signals from tyrosine kinase receptors and small GTPases. Specifically, WASF1:

• Promotes actin filament formation and polymerization

• Participates in the WAVE complex that regulates lamellipodia formation

• Regulates actin filament reorganization through interaction with the Arp2/3 complex

• Functions in BDNF-NTRK2 endocytic trafficking and signaling from early endosomes

• Contributes to the regulation of mitochondrial dynamics

In neurons, WASF1 colocalizes with activated NTRK2 after BDNF addition in endocytic sites through association with TMEM108, and is highly expressed in brain tissue with lower expression in other tissues including testis, ovary, colon, kidney, pancreas, thymus, small intestine, and peripheral blood .

What is the significance of Tyr125 phosphorylation in WASF1 function?

Phosphorylation at Tyr125 is a critical regulatory mechanism for WASF1 activity. This post-translational modification:

• Enhances WASF1 binding to the Arp2/3 complex

• Is required for WASF1 inhibition of Arp2/3-mediated stress fiber formation

• Alters WASF1's ability to regulate actin dynamics during cell migration and membrane ruffling

• Serves as a molecular switch in response to upstream signaling events

This specific phosphorylation site is distinct from phosphorylation of WAVE2 at Tyr-150 by Abl, which has different functional consequences in actin dynamics . The site-specific tyrosine phosphorylation thus provides precision in controlling specific activities of WAVE proteins.

Which kinases and phosphatases regulate WASF1 Tyr125 phosphorylation?

WASF1 Tyr125 phosphorylation is regulated by specific kinases and phosphatases:

In experimental contexts, researchers often use pervanadate treatment in cell cultures to increase detectable levels of phosphorylated WASF1 by inhibiting phosphatases .

What are the key properties of commercially available Phospho-WASF1 (Tyr125) antibodies?

Most commercially available Phospho-WASF1 (Tyr125) antibodies share these characteristics:

These antibodies are typically generated against synthetic phosphopeptides derived from the region surrounding Tyr125 in human WASF1, with the immunogen region often spanning amino acids 91-140 .

How can I validate the specificity of a Phospho-WASF1 (Tyr125) antibody?

To ensure your antibody specifically detects phosphorylated WASF1 at Tyr125, employ these validation methods:

• Phosphatase treatment control: Split your samples and treat one set with phosphatase to remove phosphorylation. The antibody signal should disappear or significantly decrease.

• Blocking peptide competition: Pre-incubate the antibody with:

  • Phospho-WAVE1 (Tyr125) peptide - should block signal

  • Non-phosphorylated WAVE1 (Tyr125) peptide - should not block signal

  • Phospho-WAVE1 (Tyr150) peptide - should not block signal

• Cross-reactivity assessment: Test against samples containing WAVE2 (phosphorylated at Tyr-124) and WAVE3 (phosphorylated at Tyr-125) to ensure specificity .

• Kinase manipulation: Compare samples from cells with activated Src kinase versus control cells. The signal should be stronger in Src-activated samples .

• Expected molecular weight verification: Confirm detection of an 80 kDa band (observed molecular weight) or 62-70 kDa band (calculated molecular weight) in Western blot applications .

As noted in the literature, high-quality antibodies like those in search result have been cross-adsorbed to phospho-WAVE (Tyr-150) and unphosphorylated WAVE (Tyr-125) peptides before affinity purification to improve specificity.

What are the recommended dilutions and conditions for using Phospho-WASF1 (Tyr125) antibody in various applications?

The optimal working conditions vary by application:

For cell-based ELISA applications, specialized kits are available that include controls for normalization such as GAPDH antibodies and Crystal Violet whole-cell staining .

What sample preparation methods are recommended for optimal results?

For Western blotting:

• Use fresh samples when possible

• Include phosphatase inhibitors in lysis buffers to preserve phosphorylation status

• Consider using pervanadate treatment (for cell cultures) to increase phosphorylation signal

• Prepare samples in buffer containing 50% glycerol, 0.5% BSA, and 0.02% sodium azide for stability

For immunohistochemistry:

• Use formalin/PFA-fixed paraffin-embedded sections

• Consider antigen retrieval methods to expose the phosphorylation site

• Block thoroughly to reduce background

For cell-based assays:

• Cell-based ELISA kits enable detection of WASF1 phosphorylation without the need for cell lysis

• These can be used for screening effects of treatments, inhibitors, or activators on WASF1 phosphorylation

How can I troubleshoot weak or nonspecific signals?

If encountering issues with antibody performance:

• For weak signals:

  • Increase antibody concentration (while staying within recommended range)

  • Extend incubation time

  • Use signal enhancement systems compatible with your detection method

  • Ensure samples contain activated Src kinase or treat with phosphatase inhibitors to increase phosphorylated protein content

• For nonspecific signals:

  • Increase blocking time and concentration

  • Try different blocking agents (BSA, normal serum, commercial blockers)

  • Perform additional washing steps

  • Confirm antibody specificity using the validation methods described in section 2.2

  • Consider the cross-reactivity noted in some antibodies with similar regions in WAVE2 (Tyr-124) and WAVE3 (Tyr-125)

• For sample degradation issues:

  • Use fresh samples when possible

  • Include protease and phosphatase inhibitors in all buffers

  • Store antibodies as recommended (typically at -20°C)

  • Avoid repeated freeze/thaw cycles

How can Phospho-WASF1 (Tyr125) antibody be used to study actin cytoskeleton dynamics?

Phospho-WASF1 (Tyr125) antibodies enable researchers to investigate the molecular mechanisms of actin cytoskeleton regulation by:

• Visualizing active WASF1 localization: Using immunofluorescence to track where phosphorylated WASF1 accumulates during cellular processes such as membrane ruffling, lamellipodia formation, and cell migration.

• Temporal activation studies: Monitoring the timing of WASF1 phosphorylation following stimulation with growth factors or activation of Rac GTPase.

• Structure-function relationship analysis: Comparing the distribution and activity of phosphorylated versus non-phosphorylated WASF1 to elucidate how this modification affects:

  • Binding to the Arp2/3 complex

  • Inhibition of Arp2/3-mediated stress fiber formation

  • Regulation of lamellipodia formation

• Signaling pathway elucidation: Investigating how WASF1 connects Rac activation to Arp2/3-mediated actin polymerization .

The Phospho-WASF1 (Tyr125) antibody can be particularly valuable in revealing the dot-like pattern of WASF1 in the cytoplasm and its concentration in Rac-regulated membrane-ruffling areas .

What is known about the differential regulation of WASF1 Tyr125 phosphorylation in neuronal versus non-neuronal cells?

Research using Phospho-WASF1 (Tyr125) antibodies has revealed distinctive patterns of regulation:

In neuronal cells:

• WASF1 is highly expressed in brain tissue

• In neurons, phosphorylated WASF1 colocalizes with activated NTRK2 after BDNF addition

• This colocalization occurs in endocytic sites through association with TMEM108

• WASF1 is required for BDNF-NTRK2 endocytic trafficking and signaling from early endosomes

In non-neuronal cells:

• Src phosphorylation of WAVE1 at Tyr-125 enhances binding to the Arp2/3 complex

• This phosphorylation is required for WAVE inhibition of Arp2/3-mediated stress fiber formation

• In fibroblasts, by contrast, WAVE2 phosphorylation at Tyr-150 by Abl may enhance Arp2/3 complex actin nucleation and microspike formation

These differences highlight that site-specific tyrosine phosphorylation provides precise control of WAVE protein activities in different cellular contexts.

How might WASF1 Tyr125 phosphorylation be implicated in disease mechanisms?

While direct evidence linking WASF1 Tyr125 phosphorylation to specific diseases is still emerging, the functional role of WASF1 suggests several potential pathological connections:

• Neurological disorders: Given WASF1's high expression in brain tissue and its role in neuronal development, dysregulation of its phosphorylation could contribute to neurodevelopmental and neurodegenerative conditions.

• Cancer progression: As WASF1 regulates actin dynamics important for cell migration, aberrant phosphorylation at Tyr125 could potentially influence cancer cell invasion and metastasis by altering cytoskeletal remodeling capabilities.

• Immune system dysfunction: The relationship between WASF1 and Wiskott-Aldrich syndrome suggests that phosphorylation abnormalities might contribute to immune dysregulation .

• Synaptic pathologies: The localization of WASF1 at synaptic junctions indicates that phosphorylation at Tyr125 may affect synaptic plasticity, potentially contributing to cognitive or psychiatric disorders.

Research using phosphorylation-specific antibodies is valuable for investigating these potential disease connections by enabling:

• Comparison of phosphorylation levels between normal and pathological tissues

• Screening for compounds that modulate WASF1 phosphorylation as potential therapeutic agents

• Studying how disease-associated mutations affect WASF1 phosphorylation status

How does phosphorylation of WASF1 at Tyr125 compare to other phosphorylation sites in the WASP protein family?

The WASP protein family exhibits distinct phosphorylation patterns with different functional consequences:

These differences highlight how site-specific phosphorylation provides precise control over cytoskeletal dynamics in different cellular contexts. Antibodies that can distinguish between these closely related phosphorylation sites are crucial for studying their specific roles.

What emerging research questions could be addressed using Phospho-WASF1 (Tyr125) antibodies?

Several promising research directions could benefit from Phospho-WASF1 (Tyr125) antibodies:

• Mechanistic studies of neuronal development: Investigating how WASF1 Tyr125 phosphorylation influences neurite outgrowth, axon guidance, and synapse formation.

• Cancer cell migration and invasion: Exploring the role of WASF1 phosphorylation in tumor cell motility and metastatic potential.

• Drug discovery: Screening compounds that modulate WASF1 phosphorylation as potential therapeutic agents for cytoskeletal-related disorders.

• Interaction proteomics: Identifying proteins that preferentially interact with phosphorylated versus non-phosphorylated WASF1.

• Spatial-temporal dynamics: Using live-cell imaging with phospho-specific antibodies to track WASF1 activation in real-time during cellular processes.

• Cross-talk with other post-translational modifications: Investigating how phosphorylation at Tyr125 influences or is influenced by other modifications on WASF1.

Researchers should consider employing multiple complementary techniques alongside antibody-based detection to fully elucidate these complex biological processes.

What methodological advances might improve phosphorylation-specific detection of WASF1?

Future technical developments could enhance our ability to study WASF1 phosphorylation:

• Higher specificity antibodies: Development of monoclonal antibodies with even greater specificity for phospho-Tyr125 without cross-reactivity to similar sites in WAVE2/3.

• Improved cell-based assays: Refinement of cell-based ELISA methods to provide quantitative rather than just qualitative measurements of phosphorylation levels.

• Phospho-proteomic approaches: Integration of antibody-based detection with mass spectrometry to map all phosphorylation sites on WASF1 simultaneously.

• Biosensors: Development of FRET-based or other fluorescent biosensors to monitor WASF1 phosphorylation in living cells with high spatial and temporal resolution.

• Cryo-EM structural studies: Using phospho-specific antibodies in conjunction with structural biology approaches to determine how phosphorylation alters WASF1 conformation and interactions.

These methodological advances would significantly enhance our understanding of WASF1 regulation and function in both normal and pathological contexts.