Phospho-MYB (S11) Recombinant Monoclonal Antibody

What is Phospho-MYB (S11) Recombinant Monoclonal Antibody?

Phospho-MYB (S11) recombinant monoclonal antibody is a highly specific antibody that recognizes the human MYB protein only when phosphorylated at serine 11. This antibody is typically generated by transfecting human phospho-MYB (S11) monoclonal antibody gene-vector clones into appropriate cell lines for in vitro production, followed by purification from tissue culture supernatant (TCS) through affinity-chromatography techniques. The antibody generally has a rabbit IgG isotype and is designed to specifically detect the phosphorylated form of MYB, allowing researchers to study this post-translational modification in various experimental contexts .

The generation of these antibodies involves immunizing host animals with synthetic phosphopeptides corresponding to residues surrounding S11 of human MYB, followed by selection of monoclonal antibodies that demonstrate high specificity for the phosphorylated form while showing minimal cross-reactivity with the non-phosphorylated version .

What are the common applications of Phospho-MYB (S11) antibodies in research?

Phospho-MYB (S11) antibodies can be utilized in multiple experimental applications:

When employed in immunofluorescence experiments, these antibodies allow researchers to visualize the subcellular localization of phosphorylated MYB protein, providing insights into how phosphorylation affects protein distribution within the cell. Western blot applications enable detection of phosphorylated MYB in cell or tissue lysates, helping researchers assess how various experimental conditions might alter MYB phosphorylation levels .

What is the significance of MYB S11 phosphorylation in cellular processes?

Phosphorylation of MYB at S11 plays crucial regulatory roles in multiple cellular functions:

• DNA Binding Regulation: Constitutive phosphorylation by Casein Kinase II (CK2) at serines 11 and 12 is required for full-length c-Myb to exhibit high-affinity, specific DNA binding activity in vitro. This phosphorylation event is therefore critical for MYB's function as a transcription factor .

• Transcriptional Activity Modulation: Phosphorylation at S11 affects MYB's ability to interact with DNA, particularly modulating its binding to low-affinity sites. CK2-mediated phosphorylation at serines 11 and 12 has been shown to lower the effectiveness of c-Myb DNA binding to low-affinity sites according to research by Luscher et al. .

• Regulatory Control: Similar to what has been observed with PHR1 (another transcription factor), phosphorylation at S11 may serve as a negative regulatory mechanism. Studies have demonstrated that S11 phosphorylation can decrease transcription factor activity in certain contexts, suggesting a conserved regulatory mechanism across different transcription factor families .

The interplay between phosphorylation at S11 and other post-translational modifications contributes to the fine-tuning of MYB's biological activities in different cellular contexts.

How does phosphorylation at S11 interact with other post-translational modifications of MYB?

The functional relationship between S11 phosphorylation and other post-translational modifications of MYB represents a complex regulatory network:

• Interplay with SUMOylation: Research indicates potential crosstalk between phosphorylation and SUMOylation. While studying a different phosphorylation site (Thr486), researchers observed that phosphorylation was only detected in the non-SUMOylated form of c-Myb isolated from cells treated with hyperthermia. This suggests that phosphorylation and SUMOylation may be mutually exclusive or sequentially regulated events .

• Multi-site Phosphorylation: Similar to findings with B-Myb, where initial Cdk-dependent phosphorylation enables subsequent binding and conformational changes, MYB may undergo sequential phosphorylation events. The phosphorylation at S11 might prime the protein for additional modifications or alter its conformation to expose or mask other modification sites .

• Stress-Induced Modification Patterns: Under stress conditions, the pattern of MYB phosphorylation changes significantly. While S11 phosphorylation occurs under normal conditions, additional sites become phosphorylated during cellular stress, creating a complex modification code that likely alters MYB function in response to different cellular environments .

Researchers investigating these interactions should consider experimental designs that allow for the simultaneous detection of multiple post-translational modifications, such as sequential immunoprecipitation or mass spectrometry-based approaches.

What methodological considerations are important when using Phospho-MYB (S11) antibodies?

When designing experiments with Phospho-MYB (S11) antibodies, researchers should consider several key methodological aspects:

• Validation of Phospho-Specificity: Always verify the phospho-specificity of the antibody in your experimental system. This can be accomplished by treating samples with alkaline phosphatase, which should eliminate the signal from phospho-specific antibodies. Similar approaches have been documented with other phospho-specific antibodies, such as those against B-MyB (phospho T487) .

• Sample Preparation: For optimal results, samples should be prepared with phosphatase inhibitors (e.g., 50 mM NaF, 25 mM β-glycerol phosphate, 10 mM sodium pyrophosphate, 2 mM sodium orthovanadate) to prevent dephosphorylation during sample processing .

• Control Selection: Appropriate controls should include:

  • Non-phosphorylated protein (negative control)

  • Samples treated with specific kinase inhibitors

  • Phospho-mimetic mutants (e.g., S11D) and phospho-null mutants (e.g., S11A)

  • Cell lines with known MYB phosphorylation status

• Application-Specific Considerations:

  Application	Critical Considerations
  Western Blot	Use freshly prepared samples with phosphatase inhibitors; optimize transfer conditions for phosphorylated proteins
  Immunofluorescence	Fixation method can affect epitope accessibility; test multiple fixation protocols
  Immunoprecipitation	Pre-clear lysates thoroughly; consider using phosphatase inhibitors in all buffers
  ChIP	Crosslinking conditions may affect antibody recognition of the phospho-epitope

• Antibody Storage and Handling: Store according to manufacturer recommendations, typically at -20°C for long-term storage. After reconstitution, store at 4°C for short-term use (one month). Avoid repeated freeze-thaw cycles which can degrade antibody quality and specificity .

How can researchers study the kinases responsible for MYB S11 phosphorylation?

Several experimental approaches can be employed to identify and characterize the kinases responsible for MYB S11 phosphorylation:

• In Vitro Kinase Assays: Researchers can conduct in vitro phosphorylation assays using purified MYB protein as a substrate and candidate kinases (such as CK2, which has been implicated in S11 phosphorylation). The reaction mixture typically includes kinase buffer (e.g., 40 mM HEPES-KOH pH 7.5, 5 mM MgCl₂, 200 μM ATP, 4 mM DTT), phosphatase inhibitors, and radiolabeled ATP (γ-³²P ATP) to detect phosphorylation events .

• Phosphorylation Site Mutants: Creating S11A (phospho-null) and S11D/E (phospho-mimetic) mutants allows researchers to study the functional consequences of S11 phosphorylation. Transient expression assays in cell culture systems can reveal how these mutations affect MYB activity, as demonstrated in studies with similar transcription factors .

• Kinase Inhibitor Screens: Treating cells with specific kinase inhibitors followed by analysis of MYB S11 phosphorylation status can help identify the responsible kinase(s). This approach requires subsequent validation using more specific techniques.

• Mass Spectrometry Analysis: Tandem mass spectrometry can be used to identify phosphorylated residues in MYB isolated from cells under different conditions. This technique has been successfully employed to map phosphorylation sites in c-Myb, revealing stress-induced phosphorylation patterns .

• Proximity-Based Labeling: Techniques such as BioID or APEX2 can identify proteins in close proximity to MYB, potentially revealing kinases that physically interact with MYB in cells.

The combination of these approaches provides a comprehensive strategy for characterizing the kinases responsible for MYB S11 phosphorylation and understanding the regulatory mechanisms involved.

How does MYB phosphorylation change during cell cycle progression?

MYB phosphorylation exhibits dynamic changes throughout the cell cycle, with important functional consequences:

• S-Phase Phosphorylation: Similar to B-Myb, which is phosphorylated during S phase, MYB phosphorylation status likely changes as cells progress through the cell cycle. Studies with B-Myb have shown that phosphorylation at the end of S phase correlates with the initiation of target gene expression .

• Regulation of Transcriptional Activity: Phosphorylation-dependent changes in MYB activity can influence the expression of cell cycle-regulated genes. In the case of B-Myb, phosphorylation by Cyclin A can promote transactivation in reporter assays, suggesting a mechanistic link between cell cycle progression and MYB family transcription factor activity .

• Protein Stability Regulation: Phosphorylation may also influence MYB protein stability throughout the cell cycle. Research on B-Myb has shown that it undergoes proteasome-dependent degradation during G2 phase, which may be influenced by its phosphorylation status .

To study these cell cycle-dependent changes, researchers typically synchronize cells (e.g., using double thymidine block) and collect samples at different time points after release. Chromatin immunoprecipitation (ChIP) assays can then be performed using Phospho-MYB (S11) antibodies to assess binding to target gene promoters at different cell cycle stages .

What is the relationship between cellular stress and MYB phosphorylation patterns?

Cellular stress significantly impacts MYB phosphorylation patterns, revealing a complex stress response mechanism:

• Stress-Induced Phosphorylation Sites: Mass spectrometry analysis has revealed that phosphorylation of specific residues in c-Myb (including Thr208, Ser444, and Thr486) occurs in response to stress conditions but is not detected in cells growing under physiological conditions .

• Functional Consequences: These stress-induced phosphorylation events may alter MYB's transcriptional activity, DNA binding properties, protein-protein interactions, or subcellular localization, allowing cells to adapt to stress conditions.

• Temporal Dynamics: The timing of phosphorylation events during stress response provides insights into the sequential activation of signaling pathways. Initial phosphorylation events may trigger subsequent modifications, creating a complex regulatory network.

• Interaction with Other Modifications: Notably, phosphorylation at Thr486 was detected only in the non-SUMOylated form of c-Myb isolated from cells treated with hyperthermia, suggesting coordination between different types of post-translational modifications during stress response .

To investigate these stress-induced changes, researchers can expose cells to various stressors (e.g., heat shock, oxidative stress, DNA damage) and analyze changes in MYB phosphorylation using Phospho-MYB (S11) antibodies in combination with antibodies targeting other phosphorylation sites.

How can Phospho-MYB (S11) antibodies be used to study transcriptional regulation?

Phospho-MYB (S11) antibodies provide valuable tools for investigating transcriptional regulation mechanisms:

• Chromatin Immunoprecipitation (ChIP): These antibodies can be used to selectively immunoprecipitate the phosphorylated form of MYB bound to chromatin, allowing researchers to determine how S11 phosphorylation affects binding to specific genomic loci. This approach has been used successfully with other MYB family proteins to assess promoter occupancy during cell cycle progression .

• ChIP-seq Analysis: Combining ChIP with next-generation sequencing enables genome-wide mapping of phosphorylated MYB binding sites, providing insights into the global regulatory network controlled by this transcription factor.

• Sequential ChIP (Re-ChIP): This technique allows researchers to determine whether proteins that are modified in two different ways occupy the same region of chromatin, helping to resolve whether S11 phosphorylation coexists with other modifications on the same MYB molecule bound to DNA.

• Correlation with Gene Expression: By combining ChIP data with RNA-seq or RT-qPCR analysis of target gene expression, researchers can assess how S11 phosphorylation affects MYB's transcriptional output. Studies with B-Myb have shown that phosphorylation at the end of S phase correlates with the start of target gene expression .

• Reporter Assays: Luciferase reporter constructs containing MYB binding sites can be used to assess how S11 phosphorylation affects transcriptional activation. Comparison of wild-type MYB with phospho-null (S11A) and phospho-mimetic (S11D/E) mutants can provide mechanistic insights.

What are common pitfalls when working with Phospho-MYB (S11) antibodies and how can they be addressed?

Working with phospho-specific antibodies presents several challenges that researchers should anticipate:

• Loss of Phosphorylation During Sample Processing: Phosphorylated proteins are susceptible to dephosphorylation by endogenous phosphatases during sample preparation.

  • Solution: Include phosphatase inhibitors (e.g., 50 mM NaF, 25 mM β-glycerol phosphate, 10 mM sodium pyrophosphate, 2 mM sodium orthovanadate) in all buffers used during sample preparation .

• Cross-Reactivity with Other Phosphorylated Epitopes: Some phospho-specific antibodies may recognize similar phosphorylated motifs in other proteins.

  • Solution: Validate antibody specificity using phospho-null mutants (S11A) or by treating samples with alkaline phosphatase to remove phosphorylation, as demonstrated with other phospho-specific antibodies .

• Batch-to-Batch Variability: Different lots of the same antibody may show variations in specificity and sensitivity.

  • Solution: Test each new batch against a standard sample with known phosphorylation status and consider creating a reference standard to normalize between experiments.

• Fixation-Dependent Epitope Masking: In immunohistochemistry or immunofluorescence applications, certain fixation methods may mask the phospho-epitope.

  • Solution: Compare multiple fixation protocols (e.g., paraformaldehyde, methanol, acetone) to determine optimal conditions for epitope accessibility.

• Low Signal-to-Noise Ratio: Phosphorylation events may be transient or affect only a small fraction of the total protein pool.

  • Solution: Consider enrichment strategies such as phospho-protein enrichment columns or immunoprecipitation prior to Western blotting to increase signal.

How can researchers design experiments to study the functional significance of MYB S11 phosphorylation?

To investigate the functional importance of MYB S11 phosphorylation, researchers can employ several experimental strategies:

• Site-Directed Mutagenesis: Generate S11A (phospho-null) and S11D/E (phospho-mimetic) mutants to study the effects of constitutive non-phosphorylation or phosphorylation, respectively. These mutants can be used in various functional assays to determine how phosphorylation affects MYB activity .

• Kinase Modulation: Identify and manipulate the activity of kinases responsible for S11 phosphorylation through:

  • Pharmacological inhibition

  • siRNA/shRNA-mediated knockdown

  • CRISPR/Cas9-mediated knockout

  • Overexpression of constitutively active kinase variants

• Phosphatase Regulation: Similarly, identify and modulate phosphatases that remove the phosphate group from S11 to understand the dynamic regulation of this modification.

• Integration with Multi-Omics Approaches: Combine proteomics (to identify phosphorylation events), transcriptomics (to assess gene expression changes), and ChIP-seq (to map DNA binding) to create a comprehensive view of how S11 phosphorylation affects MYB function.

• Structure-Function Analysis: Employ structural biology techniques (X-ray crystallography, NMR, cryo-EM) to determine how S11 phosphorylation affects MYB protein conformation and interactions with DNA or protein partners.

• Temporal Dynamics: Use pulse-chase labeling or inducible systems to study the kinetics of S11 phosphorylation and its relationship to MYB activity throughout the cell cycle or in response to various stimuli.

What approaches can be used to study the intersection of MYB phosphorylation and disease mechanisms?

The role of MYB phosphorylation in disease processes, particularly cancer, can be investigated through various approaches:

• Clinical Sample Analysis: Compare S11 phosphorylation levels in normal tissues versus disease samples using immunohistochemistry with Phospho-MYB (S11) antibodies. This approach can reveal correlations between phosphorylation status and disease progression or patient outcomes.

• Disease Model Systems: Utilize cell lines derived from relevant diseases or engineered to express MYB phospho-variants to study how S11 phosphorylation affects:

  • Cell proliferation

  • Apoptosis resistance

  • Migration and invasion

  • Drug sensitivity

  • Stemness properties

• Therapeutic Targeting: Investigate whether inhibiting the kinases responsible for S11 phosphorylation could have therapeutic value in diseases where MYB activity contributes to pathology.

• Genetic Association Studies: Examine whether genetic variations that affect S11 phosphorylation (e.g., mutations in MYB that disrupt the phosphorylation site or in kinases that phosphorylate S11) are associated with disease risk or outcomes.

• Pathway Integration: Determine how S11 phosphorylation interacts with known disease-associated signaling pathways, potentially revealing new therapeutic targets or biomarkers.

By combining these approaches, researchers can develop a comprehensive understanding of how MYB S11 phosphorylation contributes to disease mechanisms and identify potential opportunities for therapeutic intervention.

What emerging technologies might enhance the study of MYB phosphorylation?

Several cutting-edge technologies show promise for advancing our understanding of MYB phosphorylation:

• Phospho-Proteomic Mass Spectrometry: High-resolution mass spectrometry techniques allow for unbiased identification and quantification of phosphorylation sites across the proteome, providing a comprehensive view of how MYB phosphorylation changes under different conditions .

• Live-Cell Imaging of Phosphorylation: Phosphorylation biosensors based on fluorescence resonance energy transfer (FRET) could enable real-time monitoring of MYB phosphorylation dynamics in living cells.

• Single-Cell Phospho-Proteomics: Emerging technologies for analyzing phosphorylation events at the single-cell level will reveal cell-to-cell variability in MYB phosphorylation and its functional consequences.

• CRISPR Base Editing: Precise modification of phosphorylation sites in endogenous MYB without disrupting the surrounding sequence could provide more physiologically relevant insights than overexpression systems.

• Spatial Proteomics: Techniques that preserve spatial information while analyzing protein modifications will help determine where within the cell MYB phosphorylation occurs and how this affects its function.

• Computational Modeling: Integration of experimental data with computational approaches will enable prediction of how phosphorylation at multiple sites collectively influences MYB function and identification of potential therapeutic targets.

These emerging technologies will provide unprecedented insights into the complex regulatory network controlling MYB function through phosphorylation and other post-translational modifications.

How might understanding MYB phosphorylation contribute to therapeutic strategies?

The detailed understanding of MYB phosphorylation mechanisms offers several potential therapeutic applications:

• Targeted Kinase Inhibition: Identification of kinases responsible for pathological phosphorylation of MYB could lead to the development of specific inhibitors as therapeutic agents.

• Disruption of Phosphorylation-Dependent Interactions: Small molecules or peptides that block the interaction between phosphorylated MYB and its binding partners could modulate MYB activity in disease contexts.

• Biomarker Development: Phospho-MYB (S11) could serve as a biomarker for disease diagnosis, prognosis, or treatment response, particularly in cancers where MYB plays a significant role.

• Combination Therapy Approaches: Understanding how MYB phosphorylation affects sensitivity to existing therapies could inform rational combination strategies to overcome resistance mechanisms.

• Gene Therapy Approaches: Delivery of phospho-null or phospho-mimetic MYB variants could potentially modulate MYB activity in a therapeutic context.

As our understanding of the molecular mechanisms controlling MYB phosphorylation continues to evolve, these insights will likely reveal new opportunities for targeting MYB-dependent processes in various diseases.