Phospho-E2F1 (Thr433) Antibody

What is the biological significance of E2F1 phosphorylation at Threonine 433?

E2F1 phosphorylation at Threonine 433 serves as a critical post-translational modification that regulates multiple aspects of E2F1 function. Research indicates that this phosphorylation site plays key roles in:

• Cell cycle regulation: TFIIH-mediated phosphorylation of E2F1 at Thr433 has been shown to trigger E2F1 degradation during S phase, functioning as a regulatory mechanism for cell cycle progression .

• Subcellular localization: Phosphorylation at Thr433 by p38β MAPK promotes cytoplasmic distribution of E2F1 in differentiated keratinocytes, whereas mutation of this residue (T433A) results in nuclear retention .

• Protein stability: The phosphorylation status of Thr433 significantly impacts E2F1 protein stability. When Thr433 is mutated to alanine, E2F1 shows markedly increased stability, particularly in differentiating keratinocytes .

• Differentiation processes: Studies demonstrate that phosphorylation at this residue may be crucial for normal keratinocyte differentiation, as cells expressing the E2F1(T433A) mutant fail to express involucrin, a marker of epidermal differentiation .

This phosphorylation event represents a mechanism by which cells can precisely control E2F1-mediated transcriptional activity in response to changing cellular conditions.

How does a Phospho-E2F1 (Thr433) antibody specifically detect phosphorylated E2F1?

Phospho-E2F1 (Thr433) antibodies achieve their specificity through a sophisticated production and purification process:

• Immunogen design: The antibodies are produced using a synthetic phosphopeptide derived from the human E2F1 sequence surrounding the Thr433 phosphorylation site. Typically, this involves a short amino acid sequence (e.g., D-L-T(p)-P-L) where the threonine is phosphorylated .

• Host immunization: Rabbits are immunized with this phosphopeptide conjugated to a carrier protein like KLH (Keyhole Limpet Hemocyanin) to enhance immunogenicity .

• Affinity purification: The antibodies undergo a two-step affinity chromatography process:

  • First, purification using the phospho-specific peptide to isolate antibodies recognizing the region

  • Then, removal of non-phospho-specific antibodies using chromatography with the corresponding non-phosphorylated peptide

• Validation: The final antibodies are validated to confirm they detect E2F1 only when phosphorylated at Threonine 433 and not the unphosphorylated form of the protein .

This rigorous production method ensures the antibody has high specificity for the phosphorylated epitope, making it valuable for studying the phosphorylation status of E2F1 in various experimental contexts.

What are the optimal applications and experimental conditions for using Phospho-E2F1 (Thr433) antibodies?

Based on manufacturer specifications and research applications, the optimal conditions for using Phospho-E2F1 (Thr433) antibodies are:

Primary Applications:

Buffer Conditions:

• Standard WB blocking: 5% BSA or non-fat dry milk in TBST

• Antibody diluent: PBS with 0.02% sodium azide and 50% glycerol, pH 7.4

Detection Systems:
Compatible secondary antibodies include:

• Goat Anti-Rabbit IgG H&L (HRP-conjugated)

• Goat Anti-Rabbit IgG H&L (AP-conjugated)

• Fluorescent-labeled secondary antibodies (FITC, etc.)

Sample Preparation Considerations:

• Cell lysis should be performed with phosphatase inhibitors to preserve phosphorylation status

• For optimal results, samples should be freshly prepared or properly stored at -80°C

• When studying cell cycle dynamics, synchronization of cells may be necessary to observe cell cycle-dependent phosphorylation patterns

Controls:

• Positive control: Extracts from cells undergoing S phase or differentiation

• Negative control: Samples treated with lambda phosphatase

• Additional control: E2F1(T433A) mutant-expressing cells

The antibody typically detects a band at approximately 60-70 kDa depending on the gel percentage and running conditions .

How can researchers validate the specificity of Phospho-E2F1 (Thr433) antibody in their experimental system?

To ensure robust and reliable results, researchers should implement multiple validation approaches:

1. Phosphatase Treatment Control:

• Split your sample into two portions

• Treat one portion with lambda phosphatase before western blotting

• The signal should disappear or significantly decrease in the phosphatase-treated sample

2. Genetic Validation:

• Utilize cells expressing E2F1(T433A) mutant as a negative control

• The antibody should not recognize this mutant form

• Compare with wild-type E2F1 overexpression as a positive control

3. Knockdown/Knockout Verification:

• Use E2F1 siRNA or CRISPR-Cas9 knockout cells

• The specific band should be absent in these samples

4. Stimulus-Response Validation:

• Test samples from experimental conditions known to alter E2F1 phosphorylation:

  • Cell cycle synchronization (G1 vs S phase)

  • Differentiating keratinocytes (with Ca²⁺ treatment)

  • DNA damage induction

• Confirm expected changes in phosphorylation levels

5. Peptide Competition Assay:

• Pre-incubate the antibody with excess phospho-peptide immunogen

• This should block specific binding and eliminate the target signal

• Non-phosphorylated peptide should have minimal effect

6. Cross-Validation with Mass Spectrometry:

• For definitive validation, immunoprecipitate E2F1 and perform phospho-site mapping by mass spectrometry

• This confirms the presence of phosphorylation at Thr433 in your detected band

7. Multiple Antibody Approach:

• When possible, use antibodies from different vendors or clones targeting the same phospho-site

• Consistent results across different antibodies strengthen confidence in specificity

Implementation of at least 3-4 of these validation approaches is recommended to establish high confidence in antibody specificity within your specific experimental system.

How does phosphorylation of E2F1 at Thr433 regulate its transcriptional activity and cell cycle function?

Phosphorylation of E2F1 at Thr433 serves as a multi-faceted regulatory mechanism impacting several aspects of E2F1 function:

Transcriptional Regulation:

• TFIIH-mediated phosphorylation at Thr433 appears to act as a trigger for E2F1 degradation during S phase, thereby modulating its transcriptional activity in a cell cycle-dependent manner .

• Thr433 phosphorylation likely affects the activation domain function, as it is positioned within this critical region of the protein .

Protein Stability Control:

• Mutation of Thr433 to alanine (preventing phosphorylation) significantly increases E2F1 stability, indicating that phosphorylation at this site promotes protein turnover .

• This provides a mechanism to limit E2F1 activity after it has initiated S phase entry, preventing inappropriate DNA replication.

Subcellular Localization:

• In differentiating keratinocytes, phosphorylation at Thr433 (along with Ser403) promotes cytoplasmic localization of E2F1 .

• The E2F1(T433A) mutant shows predominant nuclear retention, suggesting that this phosphorylation facilitates nuclear export .

• This spatial regulation contributes to dampening E2F1 transcriptional activity when cells exit the proliferative cycle.

Cell Differentiation:

• E2F1 with mutations at Thr433 interferes with normal differentiation processes, as evidenced by the failure of keratinocytes expressing E2F1(T433A) to properly express involucrin (a differentiation marker) .

• This suggests phosphorylation at Thr433 is necessary for cell differentiation programs, potentially by allowing downregulation of E2F1-mediated proliferative gene expression.

Kinase Pathways:

• Multiple kinases can phosphorylate E2F1 at Thr433:

  • TFIIH has been demonstrated to phosphorylate this site in cell extracts

  • p38β MAPK has been shown to phosphorylate Thr433 in differentiating keratinocytes

• This multi-kinase regulation allows integration of different cellular signals in controlling E2F1 function.

The cumulative evidence indicates that phosphorylation at Thr433 represents a crucial switch that helps transition cells from proliferation to differentiation by controlling E2F1 stability, localization, and activity.

What is the relationship between E2F1 Thr433 phosphorylation and cancer development or progression?

The phosphorylation status of E2F1 at Thr433 has significant implications for cancer biology, with emerging evidence supporting its role in multiple aspects of cancer development and progression:

Correlation with Clinical Parameters:

• Research examining breast cancer patients has revealed associations between phosphorylated E2F1 expression patterns and clinical outcomes .

• Specific phosphorylation profiles of E2F1 have been correlated with response to chemotherapy, suggesting potential predictive value .

Therapeutic Response Prediction:

• A significant relationship has been observed between pE2F1 (Ser337) expression and positive response to chemotherapy, while other phosphorylation sites may have different implications .

• The study suggests that a pAkt1-/pE2F1+ phenotype could indicate an opportunity to minimize chemotherapeutic options in older women with breast cancer, while a pAkt1+/pE2F1- phenotype might warrant more aggressive treatment regimens .

Integration with Oncogenic Pathways:

• E2F1 phosphorylation status shows significant associations with other key cancer-related markers:

  • Correlations with hormone receptor status (ER/PR)

  • Relationships with HER2 expression

  • Associations with triple-negative breast cancer phenotypes

Cell Cycle Dysregulation:

• Since Thr433 phosphorylation regulates E2F1 degradation during S phase , dysregulation of this phosphorylation could contribute to aberrant cell cycle progression in cancer cells.

• Impaired phosphorylation might lead to E2F1 stabilization and persistent activation of proliferative gene programs.

Differentiation and Tumor Grade:

• Given that Thr433 phosphorylation appears necessary for normal differentiation processes , altered phosphorylation may contribute to dedifferentiation phenotypes in tumors.

• This connection to differentiation status could potentially relate to tumor grade and aggressiveness.

Therapeutic Implications:

• The research suggests that assessment of E2F1 phosphorylation status, potentially in combination with other markers like Akt1 phosphorylation, could inform personalized treatment approaches:

  • Stratification of patients for chemotherapy intensity

  • Identification of patients who might benefit from alternative treatment strategies

Further exploration of E2F1 Thr433 phosphorylation status in younger women with breast cancer and triple-negative breast cancers has been specifically recommended based on initial findings , suggesting recognition of its potential clinical significance in oncology research.

How do different kinases regulate E2F1 Thr433 phosphorylation in various cellular contexts?

The phosphorylation of E2F1 at Thr433 involves multiple kinases that operate in different cellular contexts, creating a complex regulatory network:

TFIIH-Mediated Phosphorylation:

• TFIIH has been identified as responsible for E2F1 phosphorylation at Thr433 in cell extracts .

• The CDK7 subunit of TFIIH likely performs this phosphorylation.

• This phosphorylation appears linked to S phase regulation, with evidence suggesting:

  • Endogenous E2F1 interacts with p62 (a component of TFIIH) during S phase

  • This interaction leads to phosphorylation at Thr433

  • The phosphorylation triggers E2F1 degradation during S phase

p38β MAPK-Mediated Phosphorylation:

• p38β MAPK has been demonstrated to phosphorylate E2F1 at Thr433 in differentiating keratinocytes .

• This occurs in response to calcium-regulated signaling cascades that trigger activation of:

  • Protein kinase C δ and η

  • p38β MAPK

• E2F1 forms a complex with p38β MAPK primarily in differentiated keratinocytes

• This phosphorylation facilitates CRM1-mediated E2F1 nuclear export and degradation

Context-Specific Regulation:

• Cell cycle context: TFIIH-mediated phosphorylation appears dominant during cell cycle progression

• Differentiation context: p38β MAPK pathway becomes active during keratinocyte differentiation

• This dual regulation allows E2F1 activity to be controlled differently in proliferating versus differentiating cells

Experimental Approaches to Study Kinase-Specific Effects:
To differentiate between these kinase pathways, researchers can:

• Use specific kinase inhibitors:

  • CDK7 inhibitors (e.g., THZ1) to block TFIIH-mediated phosphorylation

  • p38 MAPK inhibitors (e.g., SB203580) to block p38β-mediated phosphorylation

• Employ kinase-dead dominant negatives or siRNA approaches to selectively inhibit:

  • CDK7/TFIIH components

  • p38β MAPK

• Perform in vitro kinase assays with:

  • Purified TFIIH complex

  • Recombinant active p38β MAPK

  • E2F1 substrates (wild-type and mutant)

• Use phospho-specific antibodies in combination with the above approaches to monitor site-specific phosphorylation status

This multi-kinase regulation of E2F1 Thr433 phosphorylation provides cells with flexible control mechanisms to modulate E2F1 function appropriately in different cellular states and in response to different stimuli.

What techniques can be used to study the dynamics of E2F1 Thr433 phosphorylation during the cell cycle?

To comprehensively investigate the dynamics of E2F1 Thr433 phosphorylation throughout the cell cycle, researchers can employ these sophisticated techniques:

Cell Synchronization Methods:

• Double Thymidine Block: To synchronize cells at the G1/S boundary

• Serum starvation/stimulation: To synchronize cells in G0/G1 (as used in studies examining E2F1 phosphorylation upon stimulation of serum-starved cells)

• Nocodazole treatment: To arrest cells in M phase

• CDK inhibitor treatment (e.g., RO-3306): For G2/M arrest

Time-Resolved Phosphorylation Detection:

Advanced Microscopy Approaches:

• Immunofluorescence with phospho-E2F1 (Thr433) antibody:

  • Co-stain with cell cycle markers (PCNA, cyclin A, etc.)

  • Use EdU labeling to identify S phase cells

  • Quantify nuclear vs. cytoplasmic distribution

• Live-cell imaging with phospho-specific sensors:

  • Design FRET-based sensors for E2F1 Thr433 phosphorylation

  • Monitor real-time phosphorylation during cell cycle progression

Mass Spectrometry-Based Approaches:

• SILAC or TMT labeling:

  • Synchronize cells at different cell cycle stages

  • Label proteins with isotope tags

  • Immunoprecipitate E2F1

  • Quantify phosphopeptides by MS/MS to measure Thr433 phosphorylation levels

• Parallel Reaction Monitoring (PRM):

  • Targeted MS approach for quantifying specific E2F1 phosphopeptides

  • Higher sensitivity for low-abundance modifications

Genetic Tools for Functional Analysis:

• Phosphomimetic and phospho-dead mutants:

  • T433D/E (phosphomimetic)

  • T433A (phospho-dead)

  • Evaluate effects on cell cycle progression

• Degradation kinetics assessment:

  • Cycloheximide chase experiments comparing WT vs. T433A E2F1

  • Pulse-chase labeling to measure protein half-life

  • Correlation with cell cycle phases

Cell Cycle-Specific Protein Interactions:

• Proximity ligation assay (PLA):

  • Detect E2F1 interaction with TFIIH components during specific cell cycle phases

  • Visualize phospho-E2F1 interactions with degradation machinery

• BioID or TurboID proximity labeling:

  • Identify proteins interacting with E2F1 in a cell cycle-dependent manner

  • Compare interactomes of WT vs. T433A E2F1

By combining these approaches, researchers can develop a comprehensive understanding of when E2F1 Thr433 phosphorylation occurs during the cell cycle, which kinases are responsible at different phases, and how this phosphorylation affects E2F1 function, stability, and localization throughout cell cycle progression.

How should researchers interpret contradictory findings regarding E2F1 Thr433 phosphorylation across different cell types?

When encountering contradictory findings about E2F1 Thr433 phosphorylation across different cell types, researchers should consider several important contextual factors:

Cell Type-Specific Regulatory Mechanisms:

• Different cell types exhibit unique signaling networks that may differentially regulate E2F1 phosphorylation

• Keratinocytes show p38β MAPK-dependent phosphorylation during differentiation

• Other cell types may primarily utilize TFIIH-mediated phosphorylation during S phase

• These differences reflect tissue-specific regulatory mechanisms rather than contradictory findings

Cell State Considerations:

• Proliferating vs. differentiating status dramatically affects E2F1 phosphorylation patterns

• In proliferating cells, Thr433 phosphorylation may primarily regulate S phase-specific E2F1 degradation

• In differentiating cells, the same phosphorylation may facilitate nuclear export and cytoplasmic localization

• Careful documentation of cell state is essential when comparing across studies

Analytical Framework for Resolving Contradictions:

• Methodological differences assessment:

  • Compare antibody specificities and validation methods

  • Evaluate cell lysis and phosphatase inhibitor protocols

  • Consider detection methods (WB vs. IF vs. IP-MS)

• Contextual analysis:

  • Document exact cell types, culture conditions, and cell cycle/differentiation states

  • Verify expression levels of relevant kinases (p38β MAPK, CDK7/TFIIH)

  • Examine expression of phosphatases that might counteract Thr433 phosphorylation

• Integrative hypothesis development:

  • Formulate models that accommodate seemingly contradictory findings

  • Example: "Thr433 phosphorylation occurs via TFIIH during S phase in proliferating cells but shifts to p38β MAPK-mediated phosphorylation during differentiation"

  • Test these integrated models with experiments spanning multiple cell types

Practical Approach to Resolve Contradictions:

For a specific example, consider contradictory findings between studies and :

• Study indicates TFIIH phosphorylates E2F1 at Thr433 to trigger degradation during S phase

• Study suggests p38β MAPK phosphorylates the same site in differentiating keratinocytes

To resolve this apparent contradiction:

• Examine both kinases in both cell types/states

• Use specific inhibitors for each kinase

• Perform kinase knockdowns

• Assess phosphorylation status under various conditions

• Determine if these represent parallel, complementary pathways rather than contradictory findings

The goal should be to construct a unified model that accommodates the diverse regulatory mechanisms operating in different cellular contexts rather than viewing different findings as contradictory.

What are common technical challenges when working with Phospho-E2F1 (Thr433) antibodies and how can they be addressed?

Working with phospho-specific antibodies like Phospho-E2F1 (Thr433) presents several technical challenges. Here's a comprehensive troubleshooting guide:

Challenge 1: Low or No Signal in Western Blotting

Challenge 2: High Background or Non-specific Bands

Challenge 3: Inconsistent Results Between Experiments

Challenge 4: Immunofluorescence-Specific Issues

Challenge 5: Immunoprecipitation Challenges

Advanced Validation Strategies:

• Orthogonal approach validation:

  • Compare results from WB, IF, and IP-MS

  • Consistency across methods increases confidence

• Genetic validation controls:

  • Use E2F1-T433A mutant expression as negative control

  • Use phosphatase treatment control

  • Include E2F1 knockout/knockdown samples

• Stimulation/inhibition controls:

  • p38 MAPK stimulation/inhibition in keratinocytes

  • Cell cycle synchronization/release experiments

  • TFIIH inhibition experiments

By systematically addressing these challenges, researchers can achieve reliable and reproducible results when working with Phospho-E2F1 (Thr433) antibodies across various experimental applications.

What emerging technologies could advance our understanding of E2F1 Thr433 phosphorylation dynamics and function?

Several cutting-edge technologies hold promise for deeper insights into E2F1 Thr433 phosphorylation:

Single-Cell Phosphoproteomics:

• Mass cytometry (CyTOF) with phospho-specific antibodies can profile E2F1 phosphorylation at the single-cell level alongside dozens of other markers

• Single-cell proteomics using nanoPOTS (Nanodroplet Processing in One pot for Trace Samples) could potentially detect phosphorylation changes in rare cell populations

• These approaches would reveal cell-to-cell heterogeneity in phosphorylation status within tissues

Advanced Live-Cell Imaging:

• Genetically encoded biosensors for E2F1 Thr433 phosphorylation based on phospho-binding domains coupled to FRET pairs

• Fluorescent lifetime imaging microscopy (FLIM) to detect phosphorylation events with higher sensitivity

• Lattice light-sheet microscopy for high-speed 3D imaging of phosphorylation dynamics with minimal phototoxicity

• These methods would capture real-time phosphorylation changes during cell cycle progression or differentiation

Spatially-Resolved Phosphoproteomics:

• Digital spatial profiling (DSP) to map phospho-E2F1 distribution within tissue microenvironments

• Imaging mass cytometry to visualize phosphorylation patterns at subcellular resolution in tissue sections

• These techniques would reveal how phosphorylation status varies across different microenvironments in tumors or developing tissues

CRISPR-Based Approaches:

• CRISPR activation/inhibition of kinases to precisely manipulate phosphorylation networks

• Base editing to introduce phospho-null or phosphomimetic mutations at endogenous E2F1 loci

• CRISPR screens targeting phosphatases to identify regulators of E2F1 dephosphorylation

• These genetic tools would enable more precise manipulation of E2F1 phosphorylation status

Protein Interaction Technologies:

• BioID or TurboID proximity labeling coupled with phospho-specific antibodies to identify proteins interacting specifically with phosphorylated E2F1

• Cross-linking mass spectrometry to capture structural changes induced by phosphorylation

• These methods would identify phosphorylation-dependent protein interactions

AI-Enhanced Phosphorylation Network Modeling:

• Machine learning algorithms to predict context-specific phosphorylation events

• Integration of multi-omics data to build predictive models of phosphorylation networks

• These computational approaches would help interpret complex phosphorylation data

Therapeutic Modulation Approaches:

• Small molecule or peptide-based inhibitors specifically targeting E2F1 Thr433 phosphorylation

• Proteolysis-targeting chimeras (PROTACs) selectively degrading phosphorylated E2F1

• These tools would enable precise manipulation of phospho-E2F1 levels for therapeutic applications

These emerging technologies, particularly when combined, promise to transform our understanding of how E2F1 Thr433 phosphorylation contributes to normal cellular function and disease states.

How might cross-talk between different E2F1 phosphorylation sites affect interpretation of Phospho-E2F1 (Thr433) antibody data?

The interpretation of Phospho-E2F1 (Thr433) antibody data must consider the complex interplay between multiple phosphorylation sites on E2F1:

Known E2F1 Phosphorylation Sites and Their Interactions:

Analytical Challenges Due to Cross-talk:

• Conformational Effects:

  • Phosphorylation at one site can induce conformational changes affecting accessibility of other sites

  • Thr433 epitope recognition by antibodies may be influenced by phosphorylation status of nearby residues

  • Solution: Compare results using different antibody clones that may have different sensitivities to surrounding modifications

• Sequential Phosphorylation:

  • Some sites may require priming phosphorylation at other sites

  • If Thr433 phosphorylation depends on prior phosphorylation at another site, inhibiting the priming kinase will affect Thr433 phosphorylation indirectly

  • Solution: Use phosphomimetic mutations at potential priming sites to test for sequential phosphorylation

• Competitive Phosphorylation:

  • Kinases may compete for substrate access

  • Phosphorylation at one site might prevent phosphorylation at nearby sites

  • Solution: Use in vitro kinase assays with purified components to test for competitive effects

• Antibody Cross-Reactivity Concerns:

  • Phospho-specific antibodies may have reduced specificity when multiple nearby sites are phosphorylated

  • Solution: Validate antibody specificity using synthetic peptides with different phosphorylation combinations

Experimental Approaches to Address Cross-talk:

• Phospho-proteomic mapping:

  • Mass spectrometry analysis of immunoprecipitated E2F1 to identify all phosphorylated residues

  • Quantify relative abundance of different phospho-forms

  • Identify co-occurrence patterns of multiple phosphorylations

• Multi-site mutant analysis:

  • Generate E2F1 constructs with combinations of phospho-null mutations

  • Compare effects of single vs. multiple mutations on localization, stability, and function

  • Example: T433A vs. S403A/T433A vs. S337A/T433A

• Temporal phosphorylation analysis:

  • Time-course studies after stimulation

  • Determine order of appearance of different phosphorylations

  • Use specific kinase inhibitors to block individual phosphorylation events

• Phospho-antibody arrays:

  • Use cell cycle control phospho-antibody arrays that include multiple E2F1 phospho-sites

  • Compare phosphorylation patterns across different conditions

Practical Recommendations for Data Interpretation:

• Always consider potential effects of other phosphorylation sites when interpreting Thr433 phosphorylation data

• Include controls with mutations at multiple sites to dissect individual contributions

• Use complementary detection methods (e.g., MS-based approaches alongside antibody-based methods)

• When possible, examine multiple phosphorylation sites simultaneously to reveal co-regulation patterns

• Consider using phosphatase treatment followed by in vitro kinase assays to isolate specific kinase effects