Phospho-EIF4EBP1 (T70) Antibody

What is the biological significance of 4E-BP1 phosphorylation at threonine 70?

Phosphorylation of 4E-BP1 at threonine 70 (T70) plays a critical role in the regulation of cap-dependent translation. While phosphorylation by FRAP/mTOR at the priming sites Thr37 and Thr46 does not prevent the binding of 4E-BP1 to eIF4E, it is thought to prepare 4E-BP1 for subsequent phosphorylation at Ser65 and Thr70. According to the two-step phosphorylation model, these additional phosphorylation events, particularly at Thr70, contribute to the release of 4E-BP1 from eIF4E, thereby enabling the assembly of the translation initiation complex and activation of mRNA translation. This phosphorylation is regulated in response to various cellular signals including insulin signaling and UV irradiation, making it a central node in translational control mechanisms .

How does Thr70 phosphorylation coordinate with other phosphorylation sites on 4E-BP1?

Thr70 phosphorylation works in concert with other phosphorylation sites to regulate 4E-BP1 function. While traditional models suggest that Thr37/Thr46 priming phosphorylations are prerequisites for subsequent phosphorylation at Thr70, recent research has identified populations of 4E-BP1 with Thr70 phosphorylation but lacking Thr37/Thr46 phosphorylation, particularly during mitosis. Two-dimensional gel electrophoretic analysis has revealed distinct phosphorylation patterns where some eIF4E-bound 4E-BP1 isoforms (specifically the mitotic EB-γ band) show positive signals for Ser83 and Thr70 phosphorylations without the expected Thr37/Thr46 priming phosphorylations. This indicates more complex regulatory mechanisms than previously thought, suggesting that different combinations of phosphorylation events may regulate 4E-BP1 function in context-specific manners .

What are the structural characteristics of EIF4EBP1 that make Thr70 important?

EIF4EBP1 (4E-BP1) contains several important structural motifs that influence its function and regulation. The protein features three key motifs: Motif 1 (contains the canonical YXXXLφ sequence responsible for direct eIF4E binding), Motif 2 (a proline-turn-helix segment containing the Ser65 and Thr70 phosphorylation sites), and Motif 3 (a C-terminal loop required for high-affinity association with eIF4E). Thr70 is positioned at the transition between Motif 2 and Motif 3 (70IPGVTSP84), making its phosphorylation status particularly influential on the protein's conformation and binding capabilities. Additionally, 4E-BP1 contains an N-terminal RAIP motif and a C-terminal TOS motif that participate in regulating its phosphorylation. This strategic positioning of Thr70 makes it a critical regulatory site for controlling the protein's interaction with eIF4E and subsequent effects on translation initiation .

How can Phospho-EIF4EBP1 (T70) Antibody be used to study cell cycle-dependent translation regulation?

The Phospho-EIF4EBP1 (T70) Antibody serves as a powerful tool for investigating the relationship between cell cycle progression and translational control. Research has revealed distinctive patterns of 4E-BP1 phosphorylation during mitosis compared to interphase, with particular enrichment of certain phosphorylated forms during mitotic arrest. To effectively study these cycle-dependent changes, researchers can synchronize cells using mitotic inhibitors like S-trityl-L-cysteine (STLC) and compare phosphorylation patterns between asynchronous and mitotic populations. The antibody can be employed in eIF4E pulldown assays followed by immunoblotting to detect specific phosphorylation states. Proximity ligation assays combining phospho-4E-BP1 and eIF4E detection can further reveal spatial relationships between differently phosphorylated 4E-BP1 populations and eIF4E during various cell cycle phases. These approaches have demonstrated that certain phosphorylated 4E-BP1 forms (like the EB-γ band positive for T70 but negative for T37/T46 phosphorylation) are specifically enriched during mitosis .

What are the implications of using this antibody in cancer research models?

The Phospho-EIF4EBP1 (T70) Antibody has significant applications in cancer research where dysregulation of cap-dependent translation through the mTOR pathway is a common feature. By monitoring T70 phosphorylation status, researchers can evaluate the activation state of mTOR signaling and translational control in various cancer models. This approach enables the assessment of how oncogenic mutations, therapeutic interventions, or microenvironmental conditions affect translation initiation factor regulation. When designing experiments for cancer models, researchers should consider using the antibody in multiple applications, including western blotting (at 1:1000 dilution) and immunohistochemistry (at 1:50-1:100 dilution), to comprehensively characterize 4E-BP1 phosphorylation patterns. The antibody detects endogenous levels of EIF4EBP1 only when phosphorylated at threonine 70, making it suitable for quantifying relative changes in phosphorylation status across different experimental conditions or in response to targeted therapies .

How do post-translational modifications other than phosphorylation interact with Thr70 phosphorylation status?

The interplay between Thr70 phosphorylation and other post-translational modifications creates a complex regulatory network controlling 4E-BP1 function. According to available PTM data for human 4E-BP1 (UniProt Q13541), the protein undergoes multiple modifications beyond phosphorylation. For instance, acetylation at Ser2 may influence the protein's stability or interactions, potentially affecting subsequent phosphorylation events. The following table summarizes known post-translational modifications of 4E-BP1 that may interact with Thr70 phosphorylation:

Research using the Phospho-EIF4EBP1 (T70) Antibody in combination with antibodies targeting other modifications can help elucidate how these different PTMs cooperate or compete to fine-tune translational control in various physiological and pathological conditions .

What are the optimal conditions for using Phospho-EIF4EBP1 (T70) Antibody in Western blotting?

For optimal Western blotting results with Phospho-EIF4EBP1 (T70) Antibody, researchers should adhere to the following protocol guidelines. Sample preparation is critical: cells should be lysed in a buffer containing phosphatase inhibitors to preserve the phosphorylation state of 4E-BP1. The recommended dilution for Western blotting is 1:1000, though this may require optimization based on specific experimental conditions and sample types. When resolving 4E-BP1 on SDS-PAGE, it's important to use a gel system capable of separating proteins in the 15-20 kDa range, as 4E-BP1 typically appears as multiple bands between 15-20 kDa depending on its phosphorylation status. For enhanced resolution of different phosphorylated forms, longer gel runs or gradient gels are recommended. After transfer to a membrane, blocking should be performed with 5% BSA rather than milk, as milk contains phosphoproteins that may interfere with detection. Overnight primary antibody incubation at 4°C typically yields the best results. For detection, both chemiluminescence and fluorescence-based methods are suitable, with the latter offering advantages for quantitative analysis .

How can two-dimensional gel electrophoresis enhance detection of specific phosphorylated 4E-BP1 isoforms?

Two-dimensional gel electrophoresis significantly enhances the resolution and identification of differently phosphorylated 4E-BP1 isoforms that may be indistinguishable in one-dimensional SDS-PAGE. This technique separates proteins based on both isoelectric point (pI) in the first dimension and molecular weight in the second dimension. For optimal results when analyzing 4E-BP1 phosphorylation states, the following methodology is recommended: First, use a pH gradient of 3-10 for isoelectric focusing to adequately separate the various phosphorylated forms, as each phosphate group adds a negative charge that shifts the protein toward the acidic end of the spectrum. Second, employ a high-percentage (15-18%) gel for the second dimension to properly resolve the subtle molecular weight differences between phosphorylated isoforms. After transfer, immunoblotting with the Phospho-EIF4EBP1 (T70) Antibody allows specific detection of T70-phosphorylated forms. This approach has successfully identified distinct populations of 4E-BP1 molecules, including those with T70 phosphorylation but lacking T37/T46 phosphorylation, which would not be discernible in conventional one-dimensional electrophoresis. By comparing patterns from differently treated samples (e.g., asynchronous versus mitotic cells), researchers can generate comprehensive phosphorylation profiles that reveal context-specific regulatory mechanisms .

What are the appropriate controls when using Phospho-EIF4EBP1 (T70) Antibody in experimental workflows?

Implementing proper controls is essential for accurate interpretation of results when using Phospho-EIF4EBP1 (T70) Antibody. The following control strategies should be incorporated into experimental designs:

• Positive controls: Include lysates from cells treated with known activators of the mTOR pathway (e.g., insulin or serum stimulation following starvation) to induce 4E-BP1 phosphorylation at T70.

• Negative controls: Use samples treated with mTOR inhibitors like rapamycin or Torin1, which should reduce T70 phosphorylation.

• Phosphatase controls: Treat a portion of your sample with lambda phosphatase to remove phosphorylation and confirm antibody specificity.

• Knockout/knockdown validation: When possible, include samples from 4E-BP1 knockout or knockdown systems to verify antibody specificity.

• Peptide competition: Pre-incubate the antibody with the phosphopeptide used for immunization to block specific binding.

• Total protein normalization: Always probe for total 4E-BP1 on parallel blots or after stripping and reprobing to differentiate between changes in phosphorylation versus changes in protein expression.

• Loading controls: Include detection of housekeeping proteins (e.g., GAPDH, β-actin) to ensure equal loading across samples.

These controls help distinguish specific signals from background and validate the phosphorylation-specific nature of the detected bands, particularly important given the multiple phosphorylated forms of 4E-BP1 that often appear as closely migrating bands .

How can researchers distinguish between different phosphorylated forms of 4E-BP1 in complex samples?

Distinguishing between different phosphorylated forms of 4E-BP1 in complex samples requires a multi-faceted approach. First, utilize high-resolution SDS-PAGE with extended run times to maximize separation of the multiple 4E-BP1 isoforms, which typically appear as bands between 15-20 kDa. The most hyperphosphorylated form (often designated as the γ or δ band) migrates more slowly than less phosphorylated forms (α and β bands). Second, employ phospho-specific antibodies targeting different sites in combination, including those for T37/T46, S65, T70, and S83, to create a comprehensive phosphorylation profile. Third, when analyzing eIF4E-bound versus unbound fractions, use cap-affinity purification with m7GTP-Sepharose beads to isolate eIF4E and associated proteins, followed by immunoblotting with phospho-specific antibodies. This approach has revealed that some 4E-BP1 molecules phosphorylated at T70 can still associate with eIF4E, particularly if they lack phosphorylation at other sites like S65. Fourth, for the most definitive analysis, combine these approaches with two-dimensional gel electrophoresis or phosphatase treatment of parallel samples to confirm phosphorylation status. Finally, consider using proximity ligation assays to visualize the spatial relationships between differently phosphorylated 4E-BP1 forms and eIF4E in situ .

What factors can lead to false positive or negative results when using this antibody?

Several factors can contribute to false positive or negative results when using Phospho-EIF4EBP1 (T70) Antibody, requiring careful experimental design and interpretation. Potential sources of false positives include: 1) Cross-reactivity with other phosphoproteins, particularly those containing similar phosphothreonine-containing motifs - validate by including knockout/knockdown controls; 2) Inadequate blocking - use 5% BSA rather than milk to avoid phosphoprotein interference; 3) Secondary antibody cross-reactivity - perform secondary-only controls; and 4) Sample degradation leading to non-specific bands - always include protease inhibitors in lysis buffers. Conversely, false negatives may result from: 1) Phosphatase activity during sample preparation - always include phosphatase inhibitors and process samples rapidly at cold temperatures; 2) Insufficient protein loading - check with total 4E-BP1 antibody or loading controls; 3) Suboptimal antibody dilution - titrate to determine optimal concentration for your system; 4) Inefficient transfer of low molecular weight proteins - use PVDF membranes with small pore sizes and optimize transfer conditions; and 5) Epitope masking due to protein-protein interactions - consider different lysis conditions or denaturing immunoprecipitation. When troubleshooting, systematic evaluation of each of these factors can help identify the source of unexpected results .

How does the specificity of Phospho-EIF4EBP1 (T70) Antibody compare across different species?

The cross-reactivity profile of Phospho-EIF4EBP1 (T70) Antibody varies across species, requiring careful consideration when designing experiments with non-human models. Based on available product information, most commercial antibodies against phospho-T70 4E-BP1 show confirmed reactivity with human, mouse, rat, and monkey samples. This cross-reactivity is supported by the high conservation of the amino acid sequence surrounding the T70 phosphorylation site across mammalian species. The following table summarizes the documented and predicted reactivity of various phospho-T70 antibodies:

How can proximity ligation assays be optimized for studying interactions between phosphorylated 4E-BP1 and eIF4E?

Proximity ligation assays (PLAs) offer powerful insights into the spatial relationships between specifically phosphorylated 4E-BP1 forms and eIF4E within intact cells, but require careful optimization for reliable results. To effectively implement this technique, researchers should follow these methodological guidelines: First, cell fixation must balance preservation of phospho-epitopes with maintaining protein-protein interactions; paraformaldehyde fixation (4%, 10-15 minutes) followed by gentle permeabilization (0.1% Triton X-100, 5 minutes) typically provides good results. Second, antibody selection is critical - use a combination of phospho-specific 4E-BP1 antibody (such as Phospho-EIF4EBP1 (T70)) and an eIF4E antibody raised in a different species to ensure compatibility with PLA probes. Third, appropriate controls must be included: negative controls (omitting one primary antibody), positive controls (established interacting proteins), and biological controls (treatments that enhance or disrupt the interaction). Fourth, for quantitative analysis, combine PLA with cell cycle markers or other cellular compartment markers to contextualize the observed interactions. Fifth, signal optimization may require titration of antibody concentrations (typically starting at 1:50 to 1:100 for phospho-antibodies) and adjustment of incubation times. This approach has successfully revealed different subcellular distributions of variously phosphorylated 4E-BP1 forms relative to eIF4E, particularly between mitotic and interphase cells, providing insights not achievable through biochemical methods alone .