Phospho-CBL (Y674) Antibody

What is CBL and what is the significance of Y674 phosphorylation?

CBL (Casitas B-lineage Lymphoma) is a proto-oncogene that encodes a RING finger E3 ubiquitin ligase. It functions as a negative regulator of multiple signaling pathways by mediating ubiquitination of cell surface receptors, promoting their degradation through the proteasome . CBL recognizes activated receptor tyrosine kinases (including KIT, FLT1, FGFR1, FGFR2, PDGFRA, PDGFRB, CSF1R, EPHA8, and KDR) and facilitates their ubiquitination to terminate signaling .

Phosphorylation at Y674 is one of several key regulatory tyrosine phosphorylation sites on CBL, alongside Y700, Y731, and Y774. These phosphorylation events create binding sites for specific SH2-domain containing proteins, with documented interactions between Y700 and Vav, Y731 and PI3K, and Y774 and Crk/CrkL . The specific downstream interactions and signaling consequences of Y674 phosphorylation require further investigation, but its conservation across species and the development of specific antibodies targeting this modification highlight its biological significance.

What are the recommended applications and dilutions for Phospho-CBL (Y674) Antibody?

Phospho-CBL (Y674) Antibody can be utilized in multiple experimental applications:

These dilutions serve as starting points and should be optimized for your specific experimental conditions, sample types, and detection methods . The antibody has been validated across these applications, with Western blotting and flow cytometry being particularly well-documented in the literature .

What are the key properties and storage requirements for optimal antibody performance?

For optimal performance and stability of Phospho-CBL (Y674) Antibody:

The antibody is typically provided in liquid form, and proper aliquoting upon receipt is recommended to minimize freeze-thaw cycles that can degrade antibody performance . For applications requiring conjugation to fluorochromes, metal isotopes, or enzymes, carrier-free formats are available .

How should I validate the specificity of Phospho-CBL (Y674) Antibody in my experimental system?

Rigorous validation is essential for phospho-specific antibodies. Implement these methodological approaches:

The literature demonstrates specificity validation through flow cytometric analysis where anti-phospho-CBL (Y674) antibody showed signal in pervanadate-treated Jurkat cells that was blocked by pre-incubation with phospho-peptide but not with non-phospho-peptide .

What positive and negative controls should I include when using this antibody?

Comprehensive control strategy for robust experimental design:

Positive Controls:

• Pervanadate-treated cell lysates (strong inducer of tyrosine phosphorylation)

• EGF-stimulated cells (activates pathways leading to CBL phosphorylation)

• Cells with constitutively active Src family kinases

Negative Controls:

• Untreated/unstimulated cells (baseline phosphorylation)

• Phosphatase-treated samples

• Kinase inhibitor treatment (e.g., Src or Syk inhibitors, depending on context)

• Peptide competition controls (as described above)

Loading/Technical Controls:

• Total CBL antibody (on separate blot or after stripping)

• Housekeeping proteins (GAPDH, β-actin)

• Secondary-only controls (no primary antibody)

Genetic Controls (when available):

• CBL knockout or knockdown cells

• Y674F mutant-expressing cells

Including these controls will enhance data reliability and interpretation, particularly when presenting novel findings regarding CBL phosphorylation dynamics.

What techniques can I use to induce CBL Y674 phosphorylation for experimental studies?

Several approaches can effectively induce CBL phosphorylation for experimental analysis:

• Phosphatase Inhibition:

  • Pervanadate treatment (sodium orthovanadate activated with hydrogen peroxide)

  • This approach has been documented in multiple studies for inducing phosphorylation at Y674

• Receptor Tyrosine Kinase Activation:

  • EGF stimulation (activates EGFR)

  • PDGF stimulation (activates PDGFR)

  • Other growth factors relevant to your cellular system

• Integrin Engagement:

  • Fibrinogen binding to αIIbβ3 integrin (particularly in platelet studies)

  • This activation pathway induces CBL phosphorylation through Src Family Kinase (SFK) activation

• T-cell Receptor (TCR) Stimulation:

  • Anti-CD3/CD28 antibodies

  • This pathway is relevant based on CBL's role in T-cell signaling and pathologies

• Src Family Kinase Activation:

  • Chemical activators of SFKs

  • Expression of constitutively active SFK variants

For rigorous analysis, establish a time course of activation (typically ranging from 1-60 minutes after stimulation) and include appropriate pathway activation markers as controls for successful stimulation.

How does phosphorylation at Y674 compare functionally with other CBL phosphorylation sites?

CBL contains multiple tyrosine phosphorylation sites with distinct functional implications:

The differential phosphorylation pattern creates a "signaling code" that determines which downstream pathways are activated. While Y731 phosphorylation induces PI3K recruitment and activation critical for osteoclast function, the specific signaling consequences of Y674 phosphorylation require further investigation .

A methodological approach to delineate these functions involves site-directed mutagenesis to create CBL variants where specific tyrosine residues are replaced with phenylalanine (YF mutants), preventing phosphorylation at those sites. Such genetic models (e.g., CBL YF/YF mice) have been developed and can provide valuable insights into site-specific functions .

What is the role of phosphorylated CBL in platelet function and thrombosis?

CBL phosphorylation plays significant roles in platelet signaling and function, though specific Y674 effects require further investigation:

• Signaling Cascade Activation:

  • Fibrinogen binding to αIIbβ3 integrin activates Src Family Kinases (SFKs)

  • SFK activation leads to CBL Y731 phosphorylation

  • Subsequent Syk-dependent phosphorylation of CBL residues Y700 and Y774 occurs

• Differential Kinase Dependence:

  • Inhibitor studies revealed that phosphorylation of Y731 is significantly SFK-dependent

  • Phosphorylation of Y700 and Y774 shows Syk-dependence

  • Inhibition of Syk with OXSI-2 significantly decreases phosphorylation at Y700 and Y774 but has minimal effect on Y731 phosphorylation

• Functional Outcomes:

  • CBL phosphorylation regulates outside-in signaling-mediated events

  • These events influence platelet spreading, aggregation, and clot formation

  • The interaction between phosphorylated CBL residues and specific SH2-domain containing proteins (Y700-Vav, Y731-PI3K, Y774-Crk/CrkL) mediates these functional effects

For investigating CBL's role in platelet function, established methodologies include:

• Platelet isolation from fresh blood and adjustment to specific concentrations (3×10^8 platelets/mL for spreading, 2×10^8 platelets/mL for aggregation)

• Stimulation with relevant agonists

• Analysis using aggregometry, platelet spreading assays, and phosphorylation studies

How can I optimize Phospho-CBL (Y674) Antibody for use in flow cytometry?

For robust flow cytometry results with Phospho-CBL (Y674) Antibody:

• Sample Preparation:

  • Use fresh samples whenever possible

  • Fix cells with paraformaldehyde (2-4%)

  • Permeabilize thoroughly using methanol or specialized permeabilization buffers

  • Maintain cold chain throughout processing

• Staining Protocol:

  • Starting dilution: 1:20 (optimize through titration)

  • Incubation time: 30-60 minutes at room temperature or overnight at 4°C

  • Include phosphatase inhibitors in all buffers

  • Perform thorough washing between steps

• Critical Controls:

  • Unstained cells

  • Isotype control at matching concentration

  • Positive control (pervanadate-treated cells)

  • Peptide competition controls (phospho and non-phospho peptides)

• Phosphorylation-Specific Considerations:

  • Process samples rapidly as phosphorylation states are labile

  • Consider fixation immediately after stimulation

  • Establish stimulation time-course to capture optimal phosphorylation window

• Analysis Strategies:

  • Gate on live, single cells

  • Consider co-staining with total CBL for normalization

  • Present data as fold-change relative to unstimulated cells

Flow cytometric analysis has demonstrated successful detection of phosphorylated CBL (Y674) in pervanadate-treated Jurkat cells, with specificity validated through peptide competition .

What methods can detect changes in CBL E3 ligase activity following Y674 phosphorylation?

While the direct relationship between Y674 phosphorylation and CBL's E3 ligase activity isn't explicitly detailed in the search results, these methodological approaches can be employed:

• In vitro Ubiquitination Assays:

  • Express and purify wild-type CBL, Y674F (phospho-deficient), and Y674E (phospho-mimetic) variants

  • Reconstitute ubiquitination reaction with E1, E2 (preferably UB2D3/UbcH5c), ubiquitin, ATP

  • Include known CBL substrates (e.g., purified receptor tyrosine kinase domains)

  • Analyze ubiquitin chain formation by Western blot or mass spectrometry

• Cellular Substrate Degradation:

  • Express wild-type CBL, Y674F, or Y674E in CBL-knockout cells

  • Stimulate with appropriate ligands

  • Track degradation kinetics of known CBL substrates (EGFR, PDGFR, c-Kit)

  • Monitor receptor internalization using surface biotinylation or flow cytometry

• CBL Conformational Studies:

  • Assess whether Y674 phosphorylation affects the autoinhibited conformation of CBL

  • Employ techniques like hydrogen-deuterium exchange mass spectrometry

  • Examine if phosphorylation enhances interactions between CBL's RING domain and E2 enzymes

• Proximity Ligation Assays:

  • Visualize interactions between CBL and E2 enzymes or substrates

  • Compare interaction frequencies between wild-type and phospho-mutant CBL variants

Given CBL's established role as a negative regulator of many signaling pathways through receptor ubiquitination and degradation , understanding how Y674 phosphorylation modulates this activity would provide valuable insights into signaling regulation.

How can I troubleshoot non-specific binding when using Phospho-CBL (Y674) Antibody?

When encountering non-specific binding with Phospho-CBL (Y674) Antibody, implement this systematic troubleshooting approach:

• Antibody Concentration Optimization:

  • Test serial dilutions (start with recommended range: 1:500-1:2000 for WB)

  • Higher dilutions often reduce background while maintaining specific signal

• Blocking Protocol Refinement:

  • Use 5% BSA in TBST (preferred for phospho-epitopes)

  • Avoid milk as it contains phosphatases that can dephosphorylate targets

  • Consider commercial blocking reagents specifically designed for phospho-detection

• Washing Stringency Adjustment:

  • Increase number of washes (5-6 washes of 5-10 minutes each)

  • Try higher detergent concentration (0.1-0.3% Tween-20)

  • Consider low salt vs. high salt wash buffers

• Sample Preparation Optimization:

  • Ensure complete protein denaturation for Western blotting

  • Use fresh phosphatase inhibitor cocktails in lysis buffers

  • Consider phospho-enrichment techniques for low-abundance targets

  • Centrifuge lysates at high speed to remove particulates

• Membrane Selection (for Western blot):

  • Compare PVDF vs. nitrocellulose

  • Low fluorescence membranes for fluorescent detection systems

• Specificity Verification:

  • Perform peptide competition with phosphorylated and non-phosphorylated peptides

  • Test antibody on samples with known phosphorylation status

  • Consider alternative antibody clones or vendors

• Technical Considerations:

  • Ensure all buffers and reagents are fresh

  • Verify pH of buffers is appropriate

  • Check quality of secondary antibody

The antibody was affinity-purified from rabbit antiserum by affinity-chromatography using epitope-specific immunogen, which should enhance specificity, but optimization for your specific experimental system is still critical .

How does CBL Y674 phosphorylation integrate with other post-translational modifications?

CBL undergoes multiple post-translational modifications that collectively regulate its function and interactions:

• Multiple Phosphorylation Sites:

  • Y674, Y700, Y731, and Y774 are key tyrosine phosphorylation sites

  • These sites are differentially regulated (e.g., Y731 is SFK-dependent while Y700 and Y774 are Syk-dependent in platelets)

  • Phosphorylation patterns likely create a combinatorial code determining downstream interactions

• Phosphorylation-Ubiquitination Crosstalk:

  • As an E3 ubiquitin ligase, CBL mediates ubiquitination of various substrates

  • Phosphorylation may influence CBL's ubiquitin ligase activity

  • CBL can also undergo self-ubiquitination, potentially regulated by its phosphorylation status

• Hierarchical Modification:

  • Evidence suggests sequential phosphorylation events, where certain sites must be phosphorylated before others

  • In platelets, SFK activation leads to Y731 phosphorylation, followed by Syk-dependent phosphorylation of Y700 and Y774

• Methodological Approaches to Study Interplay:

  • Mass spectrometry-based proteomics to identify modification patterns

  • Use of phospho-mimetic and phospho-deficient mutations at multiple sites

  • Sequential inhibition of kinases to establish dependency relationships

  • Correlation of modification patterns with functional outcomes

• Functional Consequences:

  • Different modification patterns likely determine which SH2-domain containing proteins are recruited

  • Y700 interacts with Vav, Y731 with PI3K, and Y774 with Crk/CrkL

  • These interactions initiate distinct downstream signaling cascades

Understanding this complex interplay requires systematic investigation using combinations of site-specific antibodies and genetic approaches to manipulate individual modifications.

How do I interpret Phospho-CBL (Y674) signal changes in disease models?

When interpreting Phospho-CBL (Y674) signaling changes in disease models, consider these analytical approaches:

• Baseline Normalization:

  • Always normalize phospho-CBL signal to total CBL levels

  • Account for potential changes in total CBL expression between disease and control states

  • Present data as phospho-to-total ratios rather than absolute phospho-signal

• Context-Specific Interpretation:

  • In cancer models: Evaluate in context of receptor tyrosine kinase signaling

  • In immune disorders: Consider T-cell receptor and cytokine signaling pathways

  • In bone disorders: Relate to osteoclast function (CBL is essential for osteoclastic bone resorption)

  • In platelet dysfunction: Analyze alongside integrin signaling components

• Multi-site Phosphorylation Analysis:

  • Compare Y674 phosphorylation with other CBL phosphorylation sites (Y700, Y731, Y774)

  • Different phosphorylation patterns may indicate activation of distinct signaling pathways

  • Correlate with activation status of upstream kinases (SFKs, Syk)

• Functional Correlation:

  • Link phosphorylation changes to functional outcomes

  • Assess receptor degradation/downregulation (CBL's primary function)

  • Measure activation of downstream signaling components

• Validation Strategies:

  • Pharmacological intervention (kinase inhibitors, phosphatase inhibitors)

  • Genetic approaches (site-directed mutants, knockdowns)

  • Ex vivo confirmation in patient-derived samples

• Disease-Specific Controls:

  • Include samples treated with disease-relevant stimuli

  • Compare with other established disease markers

  • Consider cell type-specific phosphorylation patterns

In T-cell acute lymphoblastic leukemia research, CBL pY674 antibody has been used to examine signaling pathway alterations , highlighting the utility of this antibody in disease-focused investigations.

What emerging techniques can enhance detection and functional analysis of CBL Y674 phosphorylation?

Emerging technologies offer new opportunities for investigating CBL Y674 phosphorylation:

• Proximity-Based Approaches:

  • Proximity ligation assays (PLA) to visualize Y674 phosphorylation in situ

  • BioID or APEX2 proximity labeling to identify proteins interacting with phosphorylated Y674

  • These techniques provide spatial information about phosphorylation events and interactions

• Single-Cell Analysis:

  • Mass cytometry (CyTOF) with metal-conjugated phospho-specific antibodies

  • Single-cell Western blotting

  • These approaches reveal cell-to-cell variability in phosphorylation patterns

• Phospho-Proteomics Integration:

  • Targeted mass spectrometry to quantify multiple phosphorylation sites simultaneously

  • Phospho-enrichment strategies to enhance detection of low-abundance modifications

  • These methods provide comprehensive views of phosphorylation networks

• Live-Cell Imaging:

  • Phospho-specific biosensors based on FRET technology

  • Optogenetic tools to induce phosphorylation with temporal and spatial precision

  • These approaches enable real-time monitoring of phosphorylation dynamics

• CRISPR-Based Approaches:

  • Base editing to introduce Y674F mutations with minimal off-target effects

  • CRISPR activation/interference to modulate expression of kinases/phosphatases

  • These genetic tools provide precise manipulation of phosphorylation pathways

• Structural Biology:

  • Cryo-EM studies of CBL in different phosphorylation states

  • Hydrogen-deuterium exchange mass spectrometry to detect phosphorylation-induced conformational changes

  • These techniques reveal structural consequences of Y674 phosphorylation

Combining these emerging technologies with established biochemical approaches will advance our understanding of CBL Y674 phosphorylation in normal physiology and disease contexts.

How can Phospho-CBL (Y674) Antibody be applied in clinical and translational research?

Phospho-CBL (Y674) Antibody offers significant potential for clinical and translational research applications:

• Cancer Biomarker Development:

  • CBL mutations are found in various malignancies, particularly myeloid neoplasms

  • Phosphorylation patterns may serve as biomarkers for disease progression or treatment response

  • Immunohistochemistry on patient tissues can correlate phosphorylation with clinical outcomes

• Targeted Therapy Response Prediction:

  • CBL regulates receptor tyrosine kinases targeted by many cancer therapies

  • Y674 phosphorylation status may predict response to kinase inhibitors

  • Monitoring phosphorylation changes during treatment could indicate development of resistance

• Immune Disorder Applications:

  • CBL regulates T-cell receptor signaling, relevant to autoimmune conditions

  • Phosphorylation analysis in patient-derived immune cells may reveal pathological alterations

  • Could help stratify patients for immunomodulatory therapies

• Platelet Function Assessment:

  • CBL phosphorylation plays important roles in platelet signaling

  • Could provide insights into bleeding disorders or thrombotic conditions

  • May predict response to antiplatelet therapies

• Bone Disorder Research:

  • CBL is essential for osteoclastic bone resorption

  • Phosphorylation analysis could advance understanding of metabolic bone diseases

  • Potential application in osteoporosis research and treatment monitoring

• Drug Discovery Applications:

  • Screening compounds that modulate CBL phosphorylation

  • Developing drugs targeting specific CBL-dependent pathways

  • Monitoring on-target effects of kinase inhibitors

• Methodological Considerations for Clinical Translation:

  • Standardization of sample processing to preserve phosphorylation

  • Development of clinical-grade assays with appropriate controls

  • Comparison with established biomarkers

The use of Phospho-CBL (Y674) Antibody in translational research could bridge fundamental signaling studies with clinical applications, potentially leading to new diagnostic and therapeutic approaches.