Phospho-EIF4G1 (S1148) Antibody

What is the biological significance of EIF4G1 phosphorylation at Ser1148?

EIF4G1 phosphorylation at Ser1148 plays crucial roles in several cellular processes. Most notably, it is the only phosphorylation site found in the eIF4E/eIF4G association complex isolated from cap-containing matrix (m7GTP-Sepharose) . This phosphorylation is specifically modulated in the hippocampal CA1 region, with increased levels and colocalization with eIF4E in this vulnerable brain region in response to ischemia-reperfusion (IR) stress . Research demonstrates that while phosphorylation at Ser1148 is present in both control and ischemic conditions in brain tissue, it appears to have distinctive regulatory functions during stress responses . Furthermore, studies suggest that eIF4G1 phosphorylation at Ser1148 may compete with 4E-BPs to maintain limited cap-dependent translation during apoptotic progression in vulnerable neurons .

How does Phospho-EIF4G1 (S1148) differ from other EIF4G1 phosphorylation sites?

Unlike other EIF4G1 phosphorylation sites (Ser1185 and Ser1231), phosphorylation at Ser1148 demonstrates several unique characteristics:

• Association specificity: Only phosphorylation at Ser1148 is found in the eIF4E/eIF4G association complex isolated using cap-containing matrix (m7GTP-Sepharose)

• Response to stimuli: While phosphorylation at Ser1186 (by PKCα) and Ser1232 are responsive to TPA (phorbol ester) stimulation, Ser1148 phosphorylation does not respond to TPA

• Regulatory dynamics: Under ischemic conditions, a reduction in the eIF4E/eIF4G1 complex in the CA1 region is associated with a relative increase in Ser1148 phosphorylation in the eIF4G1 bound to eIF4E, suggesting its role in maintaining residual translation during stress

• Structural implications: Ser1148 is located in the interdomain linker (IDL) region, which controls Mnk1-eIF4G binding and assumes autoinhibitory conformations that block Mnk1 binding

These differences highlight the site-specific role of Ser1148 phosphorylation in regulating translation initiation complex formation and function.

What are the optimal protocols for using Phospho-EIF4G1 (S1148) antibody in Western blotting experiments?

For optimal Western blotting with Phospho-EIF4G1 (S1148) antibodies, researchers should follow these evidence-based protocols:

Sample Preparation:

• Treat cells with appropriate stimuli (e.g., EGF at 0.1ng/ml for 30 minutes has been validated)

• Prepare whole cell lysates using RIPA buffer supplemented with phosphatase inhibitors

Protocol Steps:

• Separate proteins on SDS-PAGE (6-8% gel recommended due to the high molecular weight of EIF4G1 ~175 kDa)

• Transfer to PVDF membrane (overnight at 30V is recommended for large proteins)

• Block with 5% BSA in TBST (avoid milk as it contains phosphatases)

• Dilute antibody 1:500-1:1000 in blocking buffer

• Incubate overnight at 4°C

• Wash 3-4 times with TBST

• Incubate with appropriate secondary antibody (HRP-conjugated anti-rabbit IgG)

• Develop using enhanced chemiluminescence

Critical Controls:

• Include phosphatase-treated samples as negative controls

• Use samples from cells treated with mTOR pathway activators (insulin, serum) and inhibitors (rapamycin, LY294002)

• Include blocking peptide controls to confirm specificity

The optimal dilution should be determined experimentally, but the range of 1:500-1:1000 has been validated by multiple sources .

How can researchers effectively isolate and analyze eIF4E/eIF4G complexes containing phosphorylated eIF4G1 at S1148?

To study phosphorylated eIF4G1(S1148) within eIF4E/eIF4G complexes, researchers can use the following validated approach combining cap-affinity chromatography and immunoprecipitation:

Cap-Affinity (m7GTP-Sepharose) Protocol:

• Prepare cell/tissue lysates in buffer containing 20 mM HEPES (pH 7.4), 150 mM NaCl, 0.5% NP-40, and phosphatase/protease inhibitors

• Incubate lysates with m7GTP-Sepharose beads for 2 hours at 4°C with rotation

• Wash beads 3-4 times with lysis buffer

• Elute bound proteins with SDS sample buffer or competitively with m7GTP

• Analyze by Western blot using anti-phospho-eIF4G1(S1148) antibody

eIF4G1 Immunoprecipitation Protocol:

• Prepare lysates as above

• Pre-clear with Protein A/G beads

• Incubate lysates with anti-eIF4G1 antibody overnight at 4°C

• Add Protein A/G beads and incubate for 2 hours

• Wash extensively

• Analyze by Western blot for phospho-eIF4G1(S1148) and co-precipitating proteins (eIF4E, eIF4A, etc.)

Research has demonstrated that phospho-eIF4G1(S1148) is preferentially detected in eIF4E/eIF4G complexes isolated by cap-affinity chromatography, making this technique particularly valuable . Studies showed that while phosphorylation at Ser1185 or Ser1231 was not detected in m7GTP-bound eIF4E fractions, phospho-eIF4G1(S1148) was specifically enriched in these complexes .

What controls should be included when using Phospho-EIF4G1 (S1148) antibody in immunohistochemistry?

When performing immunohistochemistry with Phospho-EIF4G1 (S1148) antibody, the following controls are essential for result validation:

Positive Controls:

• Brain tissue sections, particularly hippocampal CA1 regions, which show robust phospho-eIF4G1(S1148) signals

• Tissues from animals subjected to ischemia-reperfusion injury, which demonstrate increased phospho-eIF4G1(S1148) levels

Negative Controls:

• Blocking peptide control: Pre-incubate antibody with phospho-peptide immunogen to verify signal specificity

• Non-phosphorylated peptide control: Compare with phospho-peptide blocking to confirm phospho-specificity

• Primary antibody omission: Replace primary antibody with same-species IgG

Experimental Conditions:

• Recommended dilution: 1:100-1:300 for paraffin-embedded sections

• Antigen retrieval: Heat-induced epitope retrieval in citrate buffer (pH 6.0)

• Signal amplification: Biotin-streptavidin systems may enhance signal detection

Research has validated these controls by demonstrating that immunohistochemical signals from phospho-eIF4G1(S1148) antibody are specifically blocked by phospho-peptide but not by non-phosphopeptide, confirming the phospho-specificity of the antibody . Additionally, studies have shown increased signal in vulnerable brain regions after ischemic insult, providing a biological validation of antibody specificity .

How does phosphorylation of eIF4G1 at S1148 relate to mTOR signaling and cap-dependent translation?

The relationship between eIF4G1 S1148 phosphorylation and mTOR signaling involves several mechanistic connections:

• mTOR-mediated phosphorylation: Research indicates that serum- and insulin-mediated phosphorylation of eIF4G1 at S1148 is mediated by mTORC1 (mTOR Complex 1), as demonstrated by studies showing that raptor-silenced cells exhibit blocked phosphorylation of eIF4G1 at S1148

• Nutrient sensing pathway: eIF4G1 phosphorylation at S1148 increases upon leucine restoration in leucine-starved cells, and this response is reduced by raptor silencing, indicating its role in the amino acid-sensing function of mTORC1

• Direct phosphorylation by mTOR: Immunoprecipitated mTORC1 complex can directly phosphorylate eIF4G1 at S1148 in a Wortmannin-sensitive manner, similar to its phosphorylation of S6K1 at T389

• Functional implications: During cellular stress like ischemia-reperfusion, a reduced eIF4E/eIF4G1 complex is associated with increased S1148 phosphorylation in the remaining complex, suggesting this phosphorylation helps maintain limited cap-dependent translation during stress conditions

• Competition with 4E-BPs: Evidence suggests that eIF4G1 phosphorylated at S1148 may compete with 4E-binding proteins (4E-BPs) to maintain residual cap-dependent translation during apoptotic progression

These findings suggest that S1148 phosphorylation serves as a regulatory mechanism linking cellular stress responses to selective translation initiation, potentially allowing certain mRNAs to be translated even under conditions where global protein synthesis is suppressed.

What is the role of phosphorylated eIF4G1(S1148) in neuronal vulnerability during ischemia-reperfusion injury?

Phosphorylated eIF4G1(S1148) plays a critical role in neuronal vulnerability during ischemia-reperfusion (IR) injury, particularly in the hippocampal CA1 region:

The data suggest that eIF4G1 phosphorylated at S1148 represents a resilient form that can compete with 4E-BPs for eIF4E binding, maintaining limited translation activity that may selectively translate mRNAs involved in the apoptotic pathway rather than survival pathways, thereby contributing to the vulnerability of CA1 neurons to ischemic injury .

How does eIF4G1 phosphorylation influence its interactions with other proteins in the translation initiation complex?

eIF4G1 phosphorylation orchestrates a complex network of protein-protein interactions within the translation initiation complex. Based on detailed research findings:

Ser1148 Phosphorylation Effects:

• eIF4E binding: Phosphorylation at S1148 is specifically found in the eIF4E/eIF4G1 association complex isolated by cap-affinity chromatography, suggesting it may stabilize or promote this interaction

• Competition with 4E-BPs: Evidence indicates phosphorylated eIF4G1(S1148) may better compete with 4E-binding proteins for eIF4E binding during stress conditions

Other Phosphorylation Sites and Their Effects on Protein Interactions:

• S1232 phosphorylation (by Erk1/2):

  • Gradually enhances Mnk1-eIF4G binding upon TPA stimulation (~6-fold increase after 2 hours)

  • Causes rapid decline in binding with eIF4A, eIF4B, and eIF3A

  • Controls eIF4B binding to the distal IDL region

• S1239 phosphorylation (by Ck2-α):

  • Influences the rate of Erk1/2 phosphorylation of eIF4G1(S1232)

  • S1239A substitution reduces and delays TPA-induced S1232 phosphorylation

  • Affects downstream interactions with eIF4B/eIF3A

• Combined phosphorylation effects:

  • The degree of S1232 phosphorylation inversely correlates with eIF4B/eIF3A binding

  • Multiple, coordinated phosphorylation events control formation of the eIF4G/eIF4A/eIF4B helicase complex and its association with eIF3

These findings demonstrate that eIF4G1 phosphorylation creates a dynamic regulatory system where different phosphorylation sites influence each other and collectively coordinate the assembly and disassembly of various components of the translation initiation complex in response to cellular signaling.

How can researchers distinguish between specific and non-specific signals when using Phospho-EIF4G1 (S1148) antibody?

Distinguishing specific from non-specific signals requires implementing several critical validation strategies:

Validation Strategies:

• Blocking peptide experiments

  • Pre-incubate antibody with phospho-peptide immunogen

  • Include parallel experiment with non-phosphorylated peptide

  • Specific signals should be eliminated by phospho-peptide but not by non-phospho-peptide

• Signal pattern analysis

  • Specific signal should appear at the expected molecular weight (~175 kDa for full-length eIF4G1)

  • Expected subcellular localization (cytoplasmic and nuclear)

• Biological validation

  • Signals should increase in response to known stimuli (e.g., ischemia-reperfusion in CA1 neurons)

  • Signals should be reduced by relevant inhibitors (e.g., mTOR inhibitors for a phosphorylation dependent on mTOR)

• Phosphatase treatment

  • Treat one sample set with lambda phosphatase before immunoblotting

  • Specific phospho-signals should be eliminated by this treatment

• Cross-validation with multiple antibodies

  • Use phospho-specific antibodies from different vendors or clones

  • Compare results with total eIF4G1 antibody to assess relative phosphorylation levels

Common Sources of Non-specific Signals:

• Cross-reactivity with other phosphorylated proteins (particularly other eIF4G family members)

• Background from secondary antibody binding

• Insufficient blocking (particularly when using 5% BSA rather than 5% milk)

• Overly sensitive detection methods causing background amplification

Researchers should also consider using knockout/knockdown controls when available, though the essential nature of eIF4G1 in many cellular processes may make complete knockout models challenging to work with.

What are the key considerations when comparing eIF4G1 phosphorylation data across different experimental models?

When comparing eIF4G1 phosphorylation data across different experimental models, researchers should consider several critical factors to ensure valid interpretations:

Methodological Considerations:

• Antibody selection and validation

  • Different antibodies may have different specificities and sensitivities

  • Confirm antibodies detect the same epitope region and have been validated in each model system

• Experimental conditions affecting phosphorylation

  • Cell/tissue lysis methods (phosphatases can rapidly dephosphorylate proteins if not properly inhibited)

  • Nutritional status of cells/animals (serum starvation, fed vs. fasting state)

  • Time points of analysis (phosphorylation events can be transient)

• Quantification methods

  • Normalization approach (total eIF4G1 vs. housekeeping proteins)

  • Signal detection method (ECL vs. fluorescence-based detection)

  • Image acquisition parameters and analysis software

Biological Variables:

• Species differences

  • Human eIF4G1 sequence differs slightly from mouse/rat, potentially affecting antibody recognition

  • Kinase/phosphatase expression levels may vary across species

• Cell/tissue type variations

  • Brain regions show differential eIF4G1 phosphorylation patterns

  • Cell lines may have altered signaling pathways compared to primary cells

• Stress conditions

  • Ischemia-reperfusion induces specific phosphorylation patterns

  • Other stresses (oxidative, ER stress, etc.) may have different effects

Data Analysis Recommendations:

• Always express phosphorylation relative to total protein levels

• Include positive and negative controls consistent across experiments

• Report detailed methodological information to enable proper cross-study comparison

• Consider direct comparison experiments when merging data from different models

• Validate key findings using complementary approaches (e.g., mass spectrometry)

Understanding these variables is essential for accurate interpretation of seemingly contradictory results across different experimental systems.

What approaches can resolve conflicting data between eIF4G1 phosphorylation state and functional outcomes in translation studies?

Resolving conflicting data between eIF4G1 phosphorylation state and functional outcomes requires multi-dimensional approaches that integrate molecular, cellular, and functional analyses:

Mechanistic Resolution Strategies:

• Temporal resolution analysis

  • Perform detailed time-course experiments to capture transient phosphorylation events

  • Correlate phosphorylation kinetics with functional outcomes at multiple time points

  • Example: While initial studies showed no TPA responsiveness of S1148 phosphorylation , longer time courses might reveal delayed responses

• Spatial resolution approaches

  • Examine subcellular localization of phosphorylated eIF4G1 using fractionation and imaging

  • Determine if phosphorylation affects protein localization or complex formation in specific compartments

  • Research shows phospho-eIF4G1(S1148) colocalizes with eIF4E in specific brain regions

• Multi-site phosphorylation analysis

  • Assess how phosphorylation at S1148 interacts with other phosphorylation sites

  • Studies show phosphorylation at one site can influence modifications at other sites

  • Use phospho-mimetic and phospho-dead mutants in combination to test hierarchical relationships

• Targeted functional assays

  • Cap-dependent vs. cap-independent translation reporter assays

  • Polysome profiling to assess translation efficiency of specific mRNAs

  • In vitro reconstitution of translation initiation with purified components

Data Integration Approaches:

• Quantitative systems analysis

  • Develop mathematical models incorporating multiple phosphorylation sites and their effects

  • Simulate outcomes based on different phosphorylation combinations

  • Test model predictions with targeted experiments

• Correlation with physiological outcomes

  • Link molecular data to cellular phenotypes (cell survival, proliferation)

  • Connect to tissue-level outcomes (e.g., neuronal vulnerability in ischemia)

  • Study knockout/knockdown models with rescue using phospho-mimetic mutants

• Address technical artifacts

  • Verify antibody specificity in each experimental system

  • Control for phosphatase activity during sample preparation

  • Standardize quantification methods across experiments

Case Example: Resolving contradictory findings between reduced eIF4E/eIF4G1 complex and increased S1148 phosphorylation in ischemic CA1 neurons required integrating protein complex analysis with functional translation assays and histological outcomes to determine that the phosphorylation likely maintains a small pool of active translation machinery for specific mRNAs during apoptotic progression.

How can researchers effectively study the impact of eIF4G1 phosphorylation on specialized translation during stress conditions?

To effectively study how eIF4G1 phosphorylation affects specialized translation during stress, researchers should employ multi-layered approaches that connect molecular modifications to specific mRNA translation outcomes:

Experimental Approaches:

• Polysome profiling with RNA-seq

  • Fractionate polysomes from stressed and non-stressed cells/tissues

  • Perform RNA-seq on different fractions to identify mRNAs with altered translation efficiency

  • Connect changes to eIF4G1 phosphorylation status using parallel Western blots

  • Studies show ischemia affects translation of specific mRNAs despite general inhibition

• Ribosome profiling (Ribo-seq)

  • Provide nucleotide-resolution maps of ribosome positioning on mRNAs

  • Compare with total mRNA levels to calculate translation efficiency

  • Correlate with eIF4G1 phosphorylation state during stress

  • Research demonstrates eIF4G1 affects translation of specific mRNAs including those for mitochondrial proteins

• Phospho-specific protein complex isolation

  • Use phospho-eIF4G1(S1148) antibodies for immunoprecipitation

  • Identify associated mRNAs by RNA-seq

  • Compare bound mRNAs under different stress conditions

  • Studies show phospho-eIF4G1(S1148) is specifically found in cap-binding complexes

• SILAC or TMT-based proteomics

  • Quantify newly synthesized proteins during stress

  • Compare wild-type with eIF4G1 phospho-mutant cells

  • Identify proteins whose synthesis depends on eIF4G1 phosphorylation

Stress Models with Validated eIF4G1 Phosphorylation Responses:

• Ischemia-reperfusion models

  • Established to modulate eIF4G1 S1148 phosphorylation

  • Compare vulnerable (CA1) vs. resistant (cortical) brain regions

  • Focus on delayed neuronal death mechanisms

• Nutrient deprivation and restoration

  • Serum/insulin withdrawal and readdition modulates S1148 phosphorylation

  • Amino acid (leucine) deprivation affects mTOR-dependent phosphorylation

  • Study specialized translation during recovery phases

• Mitochondrial stress conditions

  • eIF4G1 influences mitochondrial protein synthesis and energy metabolism

  • Examine translation of nuclear-encoded mitochondrial proteins during stress

  • Connect to outcomes like mitochondrial membrane potential and ATP production

Data Integration Framework:

• Create a comprehensive map linking specific stressors → eIF4G1 phosphorylation changes → altered mRNA translation → functional outcomes

• Use phospho-mimetic and phospho-dead eIF4G1 mutants to establish causality

• Validate key findings across multiple cell types and stress models

• Connect molecular mechanisms to physiological outcomes (e.g., neuronal survival, mitochondrial function)

This integrated approach allows researchers to decipher how eIF4G1 phosphorylation serves as a regulatory hub to orchestrate selective translation during stress conditions.

What are the emerging techniques for studying eIF4G1 phosphorylation dynamics in living cells?

Emerging techniques for studying eIF4G1 phosphorylation dynamics in living cells offer unprecedented temporal and spatial resolution:

Real-time Phosphorylation Monitoring:

• Genetically encoded phosphorylation sensors

  • FRET-based sensors incorporating eIF4G1 phosphorylation sites

  • Allows real-time visualization of phosphorylation events in living cells

  • Can reveal compartment-specific phosphorylation dynamics

• Phospho-specific nanobodies

  • Develop nanobodies that specifically recognize phospho-eIF4G1(S1148)

  • Express as fluorescent fusion proteins for live imaging

  • Use for tracking phosphorylation without cell fixation

• Proximity labeling approaches

  • TurboID or APEX2 fusions to phospho-specific antibody fragments

  • Allows identification of proteins near phosphorylated eIF4G1

  • Can reveal dynamic remodeling of protein complexes

Advanced Microscopy Techniques:

• Single-molecule tracking

  • Label eIF4G1 and its interaction partners with photoactivatable fluorescent proteins

  • Track movement and colocalization in real-time

  • Measure association/dissociation kinetics at single-molecule resolution

• Super-resolution microscopy

  • Apply STORM, PALM, or STED microscopy to visualize nanoscale organization

  • Examine colocalization of phospho-eIF4G1 with translation machinery components

  • Studies already show colocalization with eIF4E in specific brain regions

• Lattice light-sheet microscopy

  • Provide rapid 3D imaging with minimal phototoxicity

  • Suitable for long-term tracking of dynamic phosphorylation events

Temporal Control Technologies:

• Optogenetic control of kinases/phosphatases

  • Light-inducible activation of kinases targeting eIF4G1

  • Allows precise temporal control of phosphorylation

  • Can be targeted to specific subcellular compartments

• Chemical-genetic approaches

  • Engineer analog-sensitive mTOR to control S1148 phosphorylation

  • Allows rapid and specific manipulation of phosphorylation state

  • Can be reversed by adding or removing chemical inducers

These emerging techniques will enable researchers to address key questions about the dynamics and functional consequences of eIF4G1 phosphorylation with unprecedented precision and temporal resolution.

How might targeting eIF4G1 phosphorylation therapeutically impact conditions like ischemic stroke or neurodegenerative diseases?

Targeting eIF4G1 phosphorylation offers promising therapeutic opportunities for ischemic stroke and neurodegenerative diseases based on emerging research:

Therapeutic Rationale:

• Ischemic stroke intervention

  • Research shows increased phospho-eIF4G1(S1148) in vulnerable CA1 neurons during ischemia-reperfusion

  • This phosphorylation may maintain translation of pro-apoptotic factors during delayed neuronal death

  • Selective inhibition could potentially preserve neurons in the penumbra region

• Neurodegenerative disease applications

  • eIF4G1 haploinsufficiency impairs hippocampus-dependent learning and memory

  • Mice with reduced eIF4G1 show defective axonal arborization and disrupted neuronal connectivity

  • Properly calibrated modulation of eIF4G1 phosphorylation might restore balanced translation

• Mitochondrial dysfunction targeting

  • eIF4G1 controls translation of mitochondrial OXPHOS proteins

  • Mitochondrial dysfunction is implicated in both stroke and neurodegeneration

  • Enhancing specific phosphorylation states might restore mitochondrial function

Potential Therapeutic Approaches:

• Small molecule modulators

  • Develop compounds that specifically inhibit or enhance S1148 phosphorylation

  • Target the relevant kinase (likely mTOR based on research)

  • Design structure-based inhibitors that prevent phospho-eIF4G1/eIF4E interaction

• Peptide-based therapeutics

  • Create cell-penetrating peptides that mimic phospho-S1148 region

  • These could competitively inhibit phospho-eIF4G1/eIF4E interaction

  • Use peptide aptamers to target specific protein-protein interactions

• mRNA-based approaches

  • Deliver modified eIF4G1 mRNAs encoding phospho-mimetic or phospho-dead mutations

  • Target delivery to affected brain regions using nanoparticles

  • Potential for transient expression with controlled duration

Therapeutic Development Considerations:

• Timing of intervention

  • Critical for stroke therapy (early vs. delayed intervention)

  • Research suggests targeting during reperfusion phase might be most effective

  • For neurodegenerative diseases, sustained low-level modulation may be required

• Cell type specificity

  • Develop neuron-targeted delivery strategies

  • Consider region-specific approaches (e.g., hippocampal targeting)

  • May require advanced blood-brain barrier penetration technologies

• Balancing translation regulation

  • Complete inhibition of eIF4G1 function would be detrimental

  • Requires precise modulation rather than complete blockade

  • Combinatorial approaches targeting multiple translation factors may be needed