Phospho-CBL (Y700) Antibody

What is the CBL protein and why is Y700 phosphorylation significant?

CBL (Casitas B-lineage lymphoma) is a proto-oncogene that encodes a RING finger E3 ubiquitin ligase. The protein mediates the transfer of ubiquitin from ubiquitin conjugating enzymes (E2) to specific substrates, targeting them for proteasome degradation . The phosphorylation of CBL at tyrosine 700 (Y700) is particularly important as it plays a crucial role in CBL activation and downstream signaling events. This specific phosphorylation serves as a critical regulatory site that influences CBL's function in cellular processes including cell growth, division, and differentiation .

When CBL is phosphorylated at Y700, it can interact with various signaling molecules and regulate complex cellular pathways. Research has demonstrated that c-CBL is phosphorylated at Y700 by several tyrosine kinases including Fyn, Yes, and Syk , indicating its importance in multiple signaling cascades.

What applications are suitable for Phospho-CBL (Y700) antibodies?

Phospho-CBL (Y700) antibodies have been validated for multiple research applications:

When designing experiments, it's important to optimize antibody concentration for your specific experimental conditions and cell/tissue types .

How should I select between polyclonal and monoclonal Phospho-CBL (Y700) antibodies?

The selection between polyclonal and monoclonal antibodies depends on your experimental goals:

Polyclonal Antibodies:

• Advantages: Recognize multiple epitopes on the phosphorylated region, potentially providing stronger signal and greater tolerance to protein denaturation or fixation

• Best for: Initial characterization studies, detection of low-abundance proteins, and applications requiring high sensitivity

• Example: Rabbit polyclonal antibodies such as CABP0780 have been validated for detecting endogenous levels of CBL when phosphorylated at Y700

Monoclonal Antibodies:

• Advantages: Provide higher specificity, better lot-to-lot consistency, and reduced background

• Best for: Quantitative studies requiring precise comparisons between samples, flow cytometry, and long-term projects

• Example: The EP2225Y clone (a rabbit recombinant monoclonal) has been extensively validated for Western blot and flow cytometry applications

Recombinant monoclonal antibodies offer additional benefits such as superior lot-to-lot consistency, continuous supply, and animal-free manufacturing processes .

What positive controls should I use when validating Phospho-CBL (Y700) antibody performance?

When validating phospho-specific antibodies, appropriate controls are essential:

• Positive Cell Lines: Jurkat (human T cell leukemia) cells are the most commonly used positive control for Phospho-CBL (Y700) antibodies . These cells display detectable levels of phosphorylated CBL at Y700, especially when treated with pervanadate.

• Phosphatase Treatment Controls: Treatment of cell lysates with alkaline phosphatase serves as an excellent negative control that confirms antibody specificity for the phosphorylated form rather than total CBL protein .

• Stimulation Protocols: Treatment of cells with pervanadate (1mM for 30 minutes) significantly increases phosphorylation at Y700, providing a robust positive control . This treatment inhibits protein tyrosine phosphatases, resulting in enhanced tyrosine phosphorylation.

• Molecular Weight Verification: The phosphorylated CBL protein typically appears at approximately 120 kDa on Western blots, though the calculated molecular weight is around 100 kDa . This discrepancy is likely due to post-translational modifications.

How does CBL Y700 phosphorylation participate in PI3K/AKT signaling pathways?

CBL Y700 phosphorylation serves as a critical regulatory node in PI3K/AKT signaling:

• Activation Mechanism: Phosphorylation at Y700 creates a binding site for signaling proteins, particularly Vav (a hematopoietic-restricted Rac guanine nucleotide exchange factor), which undergoes c-CBL-dependent ubiquitination upon recruitment to phospho-Y700 .

• CBL Mutations and PI3K/AKT Signaling: Research has shown that CBL mutations lead to increased CBL phosphorylation at Y700 and Y774 (known LYN target sites), which correlates with enhanced PI3K/AKT pathway activation . Western blot analyses have confirmed increased phosphorylation of AKT at S473 and ribosomal S6 at S235/236 in CBL-mutant cells compared to wild-type cells .

• Differential Regulation: While Y700 phosphorylation is primarily Syk-dependent, it functions in concert with other phosphorylation sites (particularly Y731) to coordinate downstream signaling cascades . This interconnected regulation ensures precise control of cellular responses.

• Functional Outcomes: Enhanced PI3K/AKT signaling through CBL Y700 phosphorylation influences cell proliferation, survival, and metabolism, with important implications for normal cellular function and disease states, particularly in hematopoietic cells .

What is the role of CBL Y700 phosphorylation in platelet functional responses?

CBL Y700 phosphorylation plays specific roles in platelet function:

• Integrin-Mediated Phosphorylation: Upon platelet adhesion to immobilized fibrinogen, c-CBL undergoes phosphorylation at Y700, Y731, and Y774. This phosphorylation is initiated by integrin αIIbβ3 engagement and outside-in signaling .

• Differential Kinase Dependencies: While all three phosphorylation sites are affected by pan-Src family kinase (SFK) inhibitors like PP2, Y700 and Y774 phosphorylation are more specifically dependent on Syk activity. Treatment with the Syk inhibitor OXSI-2 significantly reduces Y700 and Y774 phosphorylation without substantially affecting Y731 phosphorylation .

• Functional Implications: Knockout studies have revealed that c-CBL deficiency results in reduced platelet spreading and delayed clot retraction, indicating its importance in platelet functional responses . Studies using c-CBL YF/YF knock-in mice (with mutation at Y737, the murine equivalent of human Y731) have helped delineate the specific contributions of different phosphorylation sites to platelet function.

• Signaling Cascade: The data suggest a model where fibrinogen binding to αIIbβ3 triggers SFK activation, leading to c-CBL Y731 phosphorylation and subsequent Syk-dependent phosphorylation of Y700 and Y774 . This sequential phosphorylation regulates downstream platelet functional responses.

How can I optimize Western blot detection of phosphorylated CBL at Y700?

For optimal Western blot detection of phospho-CBL (Y700):

• Sample Preparation:

  • Rapidly lyse cells in buffer containing phosphatase inhibitors (sodium orthovanadate, sodium fluoride, and sodium pyrophosphate) to preserve phosphorylation status

  • Maintain samples at 4°C throughout processing to minimize phosphatase activity

  • For maximal phospho-signal, consider treating cells with pervanadate (1mM for 30 minutes) before lysis

• Gel Electrophoresis and Transfer:

  • Use 7-8% gels for optimal resolution of the 120kDa phospho-CBL protein

  • Consider longer transfer times (overnight at low voltage) for complete transfer of high molecular weight proteins

• Antibody Incubation:

  • Recommended dilutions range from 1:500 to 1:2000 for Western blotting

  • Block membranes with 5% non-fat dry milk in TBST (as used in validated protocols)

  • Consider overnight primary antibody incubation at 4°C for improved signal-to-noise ratio

• Detection and Validation:

  • Include both phosphorylated (pervanadate-treated) and non-phosphorylated (phosphatase-treated) controls

  • Expected molecular weight of phospho-CBL is approximately 120kDa, though calculated MW is 100kDa

  • Consider stripping and reprobing with total CBL antibody to normalize phospho-signal to total protein levels

What approaches can resolve conflicting data when studying CBL Y700 phosphorylation?

When encountering conflicting data regarding CBL Y700 phosphorylation:

• Antibody Cross-Validation:

  • Employ multiple antibodies targeting the same phospho-site from different manufacturers or clones

  • Compare polyclonal vs. monoclonal antibody results, as they may recognize slightly different epitopes

  • Verify phospho-specificity using phosphatase treatment controls

• Kinase Inhibitor Studies:

  • Use specific inhibitors of Src family kinases (SFKs) like PP2 and Syk inhibitors like OXSI-2 to confirm the kinase dependency of Y700 phosphorylation

  • These inhibitor experiments can help resolve conflicting data about the upstream regulators of CBL phosphorylation

• Genetic Validation:

  • Consider using CBL knockout models or phospho-mutant cell lines (Y700F) to definitively establish antibody specificity

  • Compare results from CBL Y700F mutants with those from Y731F or Y774F mutants to distinguish site-specific effects

• Complementary Techniques:

  • Supplement Western blot data with mass spectrometry analysis for unambiguous phospho-site identification

  • Use immunoprecipitation followed by phospho-specific Western blotting to enrich for the target protein

  • Consider proximity ligation assays (PLA) to detect interactions dependent on Y700 phosphorylation in situ

How do CBL mutations affect Y700 phosphorylation and downstream signaling in hematological disorders?

CBL mutations have significant impacts on Y700 phosphorylation and signaling in hematological disorders:

• Enhanced Phosphorylation: Studies have demonstrated that CBL mutations drive increased phosphorylation at Y700 and Y774, which are known LYN target sites . This hyperphosphorylation contributes to dysregulated signaling in hematological malignancies.

• PI3K/AKT Pathway Activation: Global proteomic analyses of CBL-mutant cells have revealed significantly increased tyrosine phosphorylation of PI3K-associated proteins. Direct measurements confirm elevated phosphorylation of AKT (at S473) and ribosomal S6 (at S235/236) in CBL-mutant cells compared to wild-type cells .

• Differential Effects of Mutations vs. Knockout: Interestingly, complete CBL knockout cells show increased LYN protein and phosphorylation but do not display the same changes in AKT and S6 phosphorylation seen in CBL-mutant cells . This suggests that mutant CBL proteins exert gain-of-function effects rather than simple loss-of-function.

• Clinical Relevance: These findings have implications for understanding diseases like acute myeloid leukemia, where CBL mutations have been identified, and Noonan syndrome-like disorder, which is caused by CBL mutations . The dysregulated Y700 phosphorylation contributes to the pathogenesis of these conditions.

What are the emerging methods for studying dynamic changes in CBL Y700 phosphorylation in live cells?

Emerging technologies are enhancing our ability to study CBL Y700 phosphorylation dynamics:

• Phospho-Specific Biosensors:

  • FRET-based biosensors designed to detect specific phosphorylation events at Y700 can provide real-time visualization of CBL activation in living cells

  • These constructs typically contain a phospho-binding domain that recognizes the phosphorylated Y700 motif, coupled with fluorescent proteins that undergo FRET upon phosphorylation

• Flow Cytometry Applications:

  • PE-conjugated phospho-CBL (Y700) antibodies enable quantitative assessment of phosphorylation levels at the single-cell level

  • This approach allows for correlation of phosphorylation status with other cellular parameters and identification of heterogeneous responses within cell populations

• Mass Cytometry (CyTOF):

  • Integration of phospho-CBL (Y700) antibodies into CyTOF panels allows simultaneous measurement of multiple phosphorylation events and pathway activation states

  • This approach is particularly valuable for understanding how CBL phosphorylation coordinates with other signaling events in complex cellular systems

• Live-Cell Imaging Combined with Optogenetics:

  • Optogenetic tools allow precise temporal control of kinase activation to trigger CBL phosphorylation

  • When combined with phospho-specific biosensors, these approaches enable unprecedented insights into the dynamics and subcellular localization of CBL Y700 phosphorylation events

These emerging methodologies promise to provide more nuanced understanding of how CBL Y700 phosphorylation is regulated in space and time, and how it contributes to normal cellular function and disease processes.

How do different commercially available Phospho-CBL (Y700) antibodies compare in performance across applications?

A comparative analysis of commercially available Phospho-CBL (Y700) antibodies reveals notable differences:

Performance considerations:

• Monoclonal antibodies like EP2225Y (ab76002) and D16D7 (#8869) generally offer higher specificity and lower background in Western blot applications

• Polyclonal antibodies may provide advantages in applications like IHC where epitope availability can be affected by fixation

• For cross-species studies, antibodies validated across multiple species (like AF2328 and A00152Y700) are preferable

• For quantitative flow cytometry, pre-conjugated formats like PE-conjugated antibodies offer convenience and consistent performance

What are the methodological differences in analyzing CBL Y700 versus Y731 and Y774 phosphorylation?

Analyzing different CBL phosphorylation sites requires consideration of their distinct properties:

• Kinase Dependencies:

  • Y700 and Y774 phosphorylation are primarily Syk-dependent, showing significant reduction when treated with Syk inhibitors like OXSI-2

  • Y731 phosphorylation is differentially regulated by Src Family Kinases (SFKs) and is less affected by Syk inhibition

  • These differences necessitate careful inhibitor selection when studying specific phosphorylation sites

• Temporal Dynamics:

  • Research in platelets suggests a sequential phosphorylation model where SFK activation leads to Y731 phosphorylation, followed by Syk-dependent phosphorylation of Y700 and Y774

  • Time-course experiments are therefore crucial when studying the relationships between these phosphorylation events

• Functional Significance:

  • Y700 phosphorylation creates binding sites for proteins like Vav, leading to ubiquitination events

  • Y731 phosphorylation (Y737 in mice) has been specifically implicated in platelet spreading, as demonstrated through studies using Y731F knock-in mice

  • Y774 often shows similar regulation patterns to Y700 but may have distinct functional outcomes

• Detection Strategies:

  • While all sites can be detected by phospho-specific antibodies, the local sequence context differs, requiring optimization of antibody conditions for each site

  • For mass spectrometry analysis, different phospho-peptides containing these sites may ionize with varying efficiencies, requiring careful quantitative controls