Phospho-EIF4EBP1 (S65) Antibody

What are the primary research applications for Phospho-EIF4EBP1 (S65) antibodies?

Phospho-EIF4EBP1 (S65) antibodies are predominantly used in these research applications:

• Western Blotting (WB): To detect and quantify the phosphorylation status of 4E-BP1 at Ser65 in cell and tissue lysates

• Immunohistochemistry (IHC): For visualizing expression and localization patterns in tissue sections

• ELISA: For quantitative assessment of phosphorylation levels

• Dot Blot: For rapid screening of samples

• Signaling Pathway Analysis: For monitoring mTOR pathway activity and response to therapeutic interventions

• Cell Cycle Studies: Particularly in examining mitosis-G1 transition regulation where CDK4 has been shown to influence 4E-BP1 phosphorylation

How can I distinguish between total 4E-BP1 and phosphorylated forms in my experiments?

To effectively differentiate between total and phosphorylated 4E-BP1:

• Sequential immunoblotting: Run duplicate gels or strip and reprobe membranes with both phospho-specific and total 4E-BP1 antibodies

• Band shift analysis: Phosphorylated 4E-BP1 demonstrates characteristic migration patterns on SDS-PAGE, appearing as multiple slower-migrating bands compared to the hypophosphorylated form

• Phosphatase treatment controls: Include samples treated with phosphatases to confirm band identity

• Signal normalization: Always normalize phospho-specific signals to total protein levels

• Phospho-mutant controls: When possible, utilize S65A mutants as negative controls to validate antibody specificity

Research has demonstrated that extensive analysis of the migration pattern of 4E-BP1 shows Ser65 phosphorylation substantially contributes to the characteristic SDS-PAGE migration pattern. The hyperphosphorylated form appears as slower-migrating bands compared to the hypophosphorylated form .

What positive and negative controls should be included when using Phospho-EIF4EBP1 (S65) antibodies?

Recommended controls for phospho-4E-BP1 (S65) experiments:

When designing phosphorylation studies, rapamycin and TOR-KI compound MLN0128 can be particularly useful as negative controls, as they have demonstrated differing effects on 4E-BP1 phosphorylation sites, with rapamycin specifically inhibiting phosphorylation at Ser65 .

How should I optimize Western blot conditions for detecting Phospho-EIF4EBP1 (S65)?

Optimization protocol for Western blotting of phospho-4E-BP1 (S65):

• Sample preparation:

  • Lyse cells in buffer containing phosphatase inhibitors (sodium fluoride, sodium orthovanadate, β-glycerophosphate)

  • Maintain cold temperatures throughout processing

  • Use fresh samples when possible or snap-freeze in liquid nitrogen

• Gel selection and separation:

  • Use 12-15% SDS-PAGE gels to optimize resolution of the low molecular weight 4E-BP1 (~12-15kDa)

  • Consider using Phos-tag™ gels for enhanced separation of phosphorylated species

• Transfer conditions:

  • Use PVDF membrane (0.2μm) rather than nitrocellulose for better protein retention

  • Short transfer times with careful temperature monitoring

• Blocking and antibody incubation:

  • 5% non-fat dry milk in TBST has been documented as effective for blocking

  • Dilute antibody according to manufacturer recommendations, typically 1:1000

  • Overnight incubation at 4°C may improve signal quality

• Signal detection considerations:

  • ECL-based detection systems with 3-minute exposure have been documented as effective

  • Be aware the antibody may detect an unknown phosphorylation-related band around 35kDa

Note that researchers should anticipate multiple bands representing different phosphorylation states, with the hyperphosphorylated forms migrating more slowly .

What tissue and cell types are most appropriate for studying EIF4EBP1 Ser65 phosphorylation?

The phosphorylation of 4E-BP1 at Ser65 can be studied across various tissue and cell types, with certain systems showing particular relevance:

Highly suitable experimental systems:

• Lymphocytes (B and T cells): Exhibit unique rapamycin sensitivity in the 4E-BP–eIF4E axis that promotes clonal expansion. Studies have shown differential phosphorylation patterns of 4E-BP1 and 4E-BP2 in activated B cells in response to rapamycin and MLN0128 .

• Cancer cell lines: Various carcinoma cell lines show overexpression of 4E-BP1 and altered phosphorylation patterns, making them valuable for studying dysregulation in cancer contexts .

• Fibroblasts: Have been used extensively for fundamental studies of 4E-BP1 phosphorylation mechanisms .

• Epidermal cells: JB6 mouse epidermal cell lines have demonstrated UVB-induced phosphorylation of 4E-BP1 at multiple sites including Ser65, mediated by p38MAPK and MSK1, providing insight into UV-promoted tumorigenesis mechanisms .

• HEK293 cells: Frequently used as model systems for studying rapamycin-sensitive phosphorylation of 4E-BP1 .

When selecting an experimental system, researchers should consider the specific signaling pathways of interest, as different cell types exhibit varying kinase activities and 4E-BP1 regulation patterns.

How can kinase-specific phosphorylation of EIF4EBP1 at Ser65 be distinguished experimentally?

Multiple kinases can phosphorylate 4E-BP1 at Ser65, including mTORC1, ERK, p38MAPK, MSK1, PIM2, and CDK4. Distinguishing between these kinases experimentally requires a multi-faceted approach:

Experimental strategies for kinase identification:

• Selective kinase inhibitors:

  • mTORC1: Rapamycin (partial inhibition) and TOR-KI compound MLN0128 (complete inhibition)

  • ERK pathway: MEK inhibitors (U0126, PD98059)

  • p38MAPK: SB203580

  • CDK4: Selective CDK4/6 inhibitors (palbociclib)

  • PIM2: PIM kinase inhibitors

• Kinase knockdown/knockout approaches:

  • siRNA or CRISPR-Cas9 targeting specific kinases

  • Genetic models with kinase deletions (e.g., 4E-BP1/2 singly deficient lymphocytes)

• In vitro kinase assays:

  • Recombinant protein substrates with single available phosphorylation sites

  • Analysis using S65[P]-specific antibodies after in vitro phosphorylation

  • Mass spectrometry to confirm site-specific modifications

• Cell cycle synchronization:

  • For CDK4-mediated phosphorylation, synchronize cells at G1/S transition

  • Compare with asynchronous populations

• Signal pathway activation:

  • UVB exposure for p38MAPK/MSK1 pathway

  • Growth factor stimulation for mTORC1 pathway

  • Ionizing radiation for ATM-dependent ERK phosphorylation

The experimental evidence indicates differential effects of kinases on 4E-BP1 phosphorylation sites. For instance, Erk efficiently phosphorylates S65 but acts less efficiently at other sites like S101 , while CDK4 has been newly identified as a 4E-BP1 kinase involved in mitosis-G1 transition .

What are the potential cross-reactivity issues with Phospho-EIF4EBP1 (S65) antibodies and how can they be addressed?

Cross-reactivity is a significant concern with phospho-specific antibodies. For Phospho-EIF4EBP1 (S65) antibodies, several important issues have been documented:

Known cross-reactivity issues:

• S101 recognition: Research has demonstrated that some anti-S65[P] antibodies also recognize 4E-BP1 phosphorylated at S101 due to sequence similarity (RNS65PV and RNS101PE contexts) .

• 4E-BP family cross-reactivity: Antibodies may recognize phosphorylated sites on other 4E-BP family members (4E-BP2, 4E-BP3) due to conserved phosphorylation motifs.

• Unknown ~35kDa band: Some antibodies recognize an unknown phosphorylation-related band around 35kDa .

Strategies to address cross-reactivity:

• Multiple mutation controls:

  • Test antibody reactivity against S65A mutants to confirm S65 specificity

  • Test against S101A mutants to assess potential S101 cross-reactivity

  • Use 3A (T36A/T45A/S65A) or 5A (T36A/T45A/S65A/T70A/S83A) mutants to fully validate specificity

• Kinase-specific phosphorylation:

  • Use Erk-mediated in vitro phosphorylation, which efficiently targets S65 but not S101

  • Compare phosphorylation patterns from different kinases

• Peptide competition assays:

  • Pre-incubate antibody with phospho-S65 peptide vs. phospho-S101 peptide

  • Differential blocking indicates cross-reactivity

• Antibody validation across sources:

  • Cross-validate using antibodies from multiple vendors

  • Note that cross-reactivity may vary between sources or batches

• Mass spectrometry confirmation:

  • For critical experiments, validate phosphorylation site assignments using phospho-proteomics

The ability of antibodies raised against S65[P] to also recognize S101[P] varies between sources and batches, making validation crucial for each experimental context .

How does the phosphorylation state of EIF4EBP1 at Ser65 correlate with other phosphorylation sites during cellular signaling?

The phosphorylation of 4E-BP1 occurs in a hierarchical and coordinated manner across multiple sites, with Ser65 playing a key role in the complete inactivation of 4E-BP1's repressor function. Understanding these relationships is crucial for interpreting experimental data:

Phosphorylation site relationships:

• Hierarchical phosphorylation model:

  • Thr37/46 phosphorylation typically precedes Ser65 phosphorylation

  • Ser65 phosphorylation often depends on prior phosphorylation at Thr37/46

  • Complete hyperphosphorylation at multiple sites (including Thr70) is required for full dissociation from eIF4E

• Differential sensitivity to inhibitors:

  • Rapamycin reduces phosphorylation of Ser65 on 4E-BP1 but has less effect on Thr37/46

  • TOR-KI compound MLN0128 suppresses phosphorylation of 4E-BP1 on both Thr36/45 and Ser65

  • This differential inhibition creates distinct phosphorylation signatures

• Cell-type specific patterns:

  • In B cells, rapamycin and MLN0128 equivalently inhibit phosphorylation of Ser65, but differentially affect Thr36/45

  • 4E-BP1 and 4E-BP2 show different phosphorylation responses to the same stimuli in lymphocytes

• Kinase-specific patterns:

  • ERK primarily phosphorylates 4E-BP1 at Ser65

  • p38MAPK and MSK1 can phosphorylate 4E-BP1 at multiple sites including Thr36, Thr45, Ser64 (human Ser65) and Thr69

  • PIM2 works in the cytoplasm to phosphorylate 4E-BP1 at Ser65

  • CDK4 has recently been identified as a 4E-BP1 kinase involved in the mitosis-G1 transition

• Migration pattern significance:

  • Extensive analysis shows that Ser65 phosphorylation substantially contributes to the SDS-PAGE migration pattern of 4E-BP1

  • The characteristic shift in migration can be used to infer phosphorylation state

Understanding these complex relationships helps researchers interpret the biological significance of observed phosphorylation patterns and design appropriate experimental controls.

What are common artifacts or misinterpretations in Phospho-EIF4EBP1 (S65) detection and how can they be avoided?

Researchers should be aware of several common artifacts and misinterpretations when working with Phospho-EIF4EBP1 (S65) antibodies:

Common issues and solutions:

• Misidentification of phosphorylation sites:

  • Issue: Antibody cross-reactivity with S101 due to sequence similarity (RNS65PV vs. RNS101PE)

  • Solution: Include S65A and S101A mutant controls; use multiple antibodies from different sources

• Misinterpretation of band patterns:

  • Issue: 4E-BP1 appears as multiple bands on Western blots representing different phosphorylation states

  • Solution: Include controls with kinase inhibitors or phosphatase treatment to identify specific forms

• Unknown bands:

  • Issue: Some antibodies recognize an unknown phosphorylation-related band around 35kDa

  • Solution: Conduct peptide competition assays and include knockout/knockdown controls

• Inconsistent results between experimental repeats:

  • Issue: Phosphorylation states are highly dynamic and sensitive to cell conditions

  • Solution: Standardize cell culture conditions, harvesting methods, and lysis buffer composition; include positive controls in each experiment

• Rapamycin resistance misinterpretation:

  • Issue: 4E-BP1 phosphorylation can become rapamycin-resistant due to PIM kinase induction

  • Solution: Use both rapamycin and catalytic mTOR inhibitors like MLN0128; assess PIM kinase activity

• Cell cycle-dependent variations:

  • Issue: 4E-BP1 phosphorylation varies throughout the cell cycle, particularly with newly discovered CDK4 involvement

  • Solution: Synchronize cells or account for cell cycle phase in interpretation

• Stimulus-dependent phosphorylation kinetics:

  • Issue: Different stimuli lead to different phosphorylation patterns and kinetics

  • Solution: Include appropriate time courses and document stimulation conditions precisely

• Degradation during sample preparation:

  • Issue: Phosphorylation signals can be lost due to phosphatase activity during processing

  • Solution: Use strong phosphatase inhibitor cocktails in all buffers; maintain samples at cold temperatures

Understanding these potential artifacts and implementing appropriate controls allows for more accurate interpretation of experimental results and avoids common pitfalls in 4E-BP1 phosphorylation analysis.

How can phosphorylation of EIF4EBP1 at Ser65 be quantified accurately across different experimental conditions?

Accurate quantification of 4E-BP1 Ser65 phosphorylation requires careful consideration of normalization approaches and quantitative techniques:

Quantification methods and considerations:

• Western blot quantification:

  • Normalization strategy: Express phospho-signal as ratio to total 4E-BP1

  • Dynamic range issues: Use appropriate exposure times to avoid signal saturation

  • Multiple band consideration: Decide whether to quantify all hyperphosphorylated bands or only specific forms

  • Software tools: Use appropriate image analysis software with background subtraction

• ELISA-based approaches:

  • Sandwich ELISA: Capture with total 4E-BP1 antibody, detect with phospho-specific antibody

  • Sensitivity enhancement: Consider amplification systems for low abundance detection

  • Standard curves: Generate using recombinant phosphorylated proteins

• Flow cytometry:

  • Single-cell resolution: Allows assessment of cell-to-cell variability in signaling

  • Multiparameter analysis: Combine with cell cycle or phenotypic markers

  • Fixation protocol: Optimize to preserve phospho-epitopes

• Mass spectrometry-based quantification:

  • Absolute quantification: Use isotope-labeled synthetic phosphopeptides as standards

  • Phosphosite occupancy: Calculate percentage of 4E-BP1 phosphorylated at Ser65

  • Multi-site analysis: Simultaneously quantify all phosphorylation sites

• In-cell western/cytoblotting:

  • High-throughput capability: Allows screening of multiple conditions

  • Direct cell-based measurement: Minimizes processing artifacts

• Normalization considerations:

  • Loading controls: Include both total 4E-BP1 and housekeeping proteins

  • Phosphorylation site ratios: Compare Ser65 to other sites (e.g., Thr37/46)

  • Treatment controls: Include maximally stimulated samples (e.g., insulin) and maximally inhibited samples (e.g., mTOR inhibitors)

• Statistical analysis:

  • Biological replicates: Minimum of three independent experiments

  • Technical replicates: Triplicate measurements when possible

  • Appropriate statistical tests: Paired analyses for treatment comparisons

Implementing these quantification strategies will provide more reliable and reproducible measurements of 4E-BP1 Ser65 phosphorylation across experimental conditions.

How should contradictory results between different detection methods for Phospho-EIF4EBP1 (S65) be interpreted?

When faced with contradictory results between different detection methods, researchers should follow a systematic approach to troubleshooting and interpretation:

Resolving contradictory results:

• Method-specific biases assessment:

  • Western blotting: May be affected by transfer efficiency, antibody specificity, and protein extraction methods

  • ELISA: Can be influenced by matrix effects and epitope accessibility in native proteins

  • Immunohistochemistry: Fixation methods significantly impact phospho-epitope detection

  • Mass spectrometry: Sample preparation and ionization efficiency can bias detection

• Antibody-related considerations:

  • Epitope differences: Different antibodies may recognize distinct conformations of phosphorylated Ser65

  • Cross-reactivity profiles: As demonstrated with S101 recognition by some S65[P] antibodies

  • Batch variation: Compare lot numbers and validate each new lot

• Biological context evaluation:

  • Cell type differences: Lymphocytes show unique responses to rapamycin in the 4E-BP–eIF4E axis

  • Pathway crosstalk: Multiple upstream kinases affect S65 phosphorylation (mTORC1, ERK, p38MAPK, PIM2, CDK4)

  • Temporal dynamics: Phosphorylation state changes rapidly upon stimulation

• Technical validation approaches:

  • Genetic controls: Use S65A mutants to confirm signal specificity

  • Pharmacological controls: Apply kinase inhibitors and phosphatase treatments

  • Method comparison: Systematically compare detection across platforms using identical samples

• Contextual interpretation framework:

  Scenario	Possible Interpretation	Recommended Investigation
  Positive WB, negative ELISA	Epitope masking in native form	Use denatured protein in ELISA
  Positive IHC, negative WB	Fixation-induced artifact	Test multiple fixation methods
  Different results with different antibodies	Epitope-specific recognition	Map epitope regions; try multiple antibodies
  Different results in different cell types	Cell-type specific regulation	Check kinase expression profiles
  Signal in WT but also in S65A mutant	Antibody cross-reactivity	Test S65A/S101A double mutant

• Resolution strategies:

  • Orthogonal validation: Confirm with an antibody-independent method (e.g., mass spectrometry)

  • Functional correlation: Assess whether observed phosphorylation correlates with expected biological outcomes (e.g., eIF4E binding)

  • Mechanistic testing: Manipulate upstream pathways to determine which accurately reflects expected biology

When interpreting contradictory results, consider that different methods may reveal complementary aspects of 4E-BP1 biology rather than simply representing technique failure.

What is the role of EIF4EBP1 Ser65 phosphorylation in cancer development and how can it be targeted therapeutically?

The phosphorylation of 4E-BP1 at Ser65 plays a critical role in cancer development through its effects on cap-dependent translation of oncogenic mRNAs:

Cancer relevance and therapeutic targeting:

• Oncogenic mechanisms:

  • Hyperphosphorylation of 4E-BP1 (including at Ser65) releases eIF4E, enabling translation of mRNAs encoding cell cycle regulators and oncoproteins

  • Overexpression of 4E-BP1 has been found in various human carcinomas

  • Phosphorylated 4E-BP1 serves as a biomarker for activated mTOR signaling in tumors

• Cancer-specific regulation:

  • UVB exposure induces phosphorylation of 4E-BP1 at multiple sites (including Ser65) via p38MAPK and MSK1, contributing to UV-promoted tumorigenesis

  • Ionizing radiation stimulates protein synthesis via ATM-dependent ERK phosphorylation of 4E-BP1 at Ser65

  • PIM kinases can directly phosphorylate 4E-BP1 at Ser65, creating rapamycin resistance in some cancers

• Therapeutic targeting strategies:

  • mTOR inhibitors: First-generation (rapamycin/rapalogs) and second-generation (TOR-KI compounds like MLN0128) targeting the mTORC1 pathway

  • Dual pathway inhibition: Combining mTOR and MEK/ERK pathway inhibitors to block multiple kinases that phosphorylate Ser65

  • CDK4/6 inhibitors: May influence 4E-BP1 phosphorylation during cell cycle progression

  • eIF4E-eIF4G interface inhibitors: Compounds like EGPI-1 target downstream of 4E-BP1 phosphorylation

• Combination therapy approaches:

  • Trametinib (MEK inhibitor) and pazopanib

  • Umbilisib and carfilzomab

  • Patamine A and silvestrol

• Biomarker development:

  • Monitoring 4E-BP1 phosphorylation status as a predictor of response to mTOR inhibitors

  • Using phospho-4E-BP1 (Ser65) levels for early disease detection and treatment formulation

• Future research directions:

  • Developing selective inhibitors of specific phosphorylation events

  • Understanding tumor-specific sensitivities to translation inhibition

  • Exploring 4E-BP1 as a target in combination therapies to improve efficacy and reduce side effects

The role of 4E-BP1 in cancer continues to be an active area of research, with particular emphasis on understanding the differential sensitivities of tumors to translation-targeting therapies and developing more effective combination approaches.

How do newly discovered kinases like CDK4 contribute to our understanding of EIF4EBP1 Ser65 phosphorylation in cell cycle regulation?

The recent identification of CDK4 as a 4E-BP1 kinase has expanded our understanding of how translation is regulated during cell cycle progression:

CDK4 and cell cycle-dependent regulation:

• Novel kinase discovery:

  • CDK4 was recently identified as a 4E-BP1 kinase through site-selective chemoproteomic methods

  • This discovery reveals a previously unknown link between cell cycle regulation and translational control

• Cell cycle phase specificity:

  • CDK4 specifically influences 4E-BP1 activity during the transition from mitosis to G1 phase

  • This timing suggests a mechanism for coordinating cell growth with cell cycle progression

  • Translation initiation may be specifically upregulated as cells enter G1 to prepare for subsequent division

• Integrated regulatory model:

  • Traditional view: mTORC1 as the primary regulator of 4E-BP1 phosphorylation

  • Expanded view: Multiple kinases including mTORC1, ERK, p38MAPK, and now CDK4 act in concert or sequentially

  • Temporal regulation: Different kinases may predominate at different cell cycle phases

• Therapeutic implications:

  • CDK4/6 inhibitors (palbociclib, ribociclib, abemaciclib) may affect translation through 4E-BP1

  • Combined targeting of cell cycle and translational machinery may provide synergistic effects

  • Resistance to mTOR inhibitors might be addressed by co-targeting CDK4/6

• Research opportunities:

  • Determining the specific 4E-BP1 phosphorylation sites targeted by CDK4

  • Investigating potential crosstalk between CDK4 and mTORC1 in regulating 4E-BP1

  • Exploring cell type-specific differences in CDK4-mediated regulation

  • Examining whether other CDKs also target 4E-BP1 at different cell cycle phases

• Experimental approaches:

  • Cell synchronization studies to isolate mitosis-G1 transition effects

  • CDK4 inhibitor time course experiments with phospho-site specific antibodies

  • In vitro kinase assays with recombinant CDK4 and 4E-BP1 variants

  • Mass spectrometry to map CDK4-dependent phosphorylation sites

The discovery of CDK4 as a 4E-BP1 kinase introduces an important new dimension to our understanding of translational control, suggesting that cap-dependent translation is specifically regulated during cell cycle transitions to coordinate growth with division.

What emerging technologies are enhancing the study of EIF4EBP1 phosphorylation dynamics in living cells?

Several cutting-edge technologies are transforming our ability to study 4E-BP1 phosphorylation with unprecedented spatial and temporal resolution:

Emerging technologies and approaches:

• Live-cell phosphorylation sensors:

  • FRET-based biosensors: Engineered constructs with 4E-BP1 sandwiched between fluorescent proteins that change energy transfer upon phosphorylation

  • Split luciferase complementation: Systems that report on 4E-BP1-eIF4E interaction in real-time

  • Phospho-specific nanobodies: Combined with fluorescent tags for real-time visualization

• Advanced microscopy techniques:

  • Super-resolution microscopy: Techniques like STORM and PALM for nanoscale visualization of 4E-BP1 localization and complex formation

  • Light-sheet microscopy: For rapid 3D imaging of phosphorylation dynamics

  • Correlative light and electron microscopy (CLEM): Connecting phosphorylation status with ultrastructural features

• Single-cell analysis technologies:

  • Single-cell phosphoproteomics: Measuring phosphorylation heterogeneity within populations

  • Mass cytometry (CyTOF): Simultaneous measurement of multiple phosphorylation sites in thousands of individual cells

  • Microfluidic approaches: Tracking individual cells over time while manipulating signaling pathways

• Genome editing for endogenous tagging:

  • CRISPR-Cas9 knock-in: Introduction of fluorescent or epitope tags at endogenous 4E-BP1 loci

  • Base editing: Precise mutation of phosphorylation sites without double-strand breaks

  • Auxin-inducible degron (AID): Rapid protein depletion systems for studying 4E-BP1 dynamics

• Optogenetic and chemogenetic tools:

  • Optogenetic kinase activation: Light-controlled activation of mTOR or other 4E-BP1 kinases

  • Chemically-induced dimerization: Rapid and reversible recruitment of 4E-BP1 to subcellular compartments

  • Photocaged phosphoamino acids: Spatiotemporal control of specific phosphorylation sites

• Computational approaches:

  • Machine learning algorithms: For pattern recognition in phosphorylation dynamics

  • Network modeling: Integrating 4E-BP1 phosphorylation into broader signaling networks

  • Single-cell trajectory analysis: Mapping phosphorylation changes through cell state transitions

• Spatial proteomics:

  • Proximity labeling: BioID or APEX2 fusion proteins to identify spatial regulators of 4E-BP1

  • Subcellular fractionation with phosphoproteomics: Compartment-specific phosphorylation analysis

These technologies are enabling researchers to move beyond static snapshots of 4E-BP1 phosphorylation to understand the dynamic regulation of translation initiation in living cells under physiological and pathological conditions.