EIF4E2 Antibody

What is EIF4E2 and how does it differ functionally from canonical eIF4E?

EIF4E2 (also termed 4EHP) is a homolog of eIF4E that binds to the 7-methylguanosine-containing mRNA 5′ cap but functions as a repressor of translation initiation rather than an activator. Unlike eIF4E, EIF4E2 cannot recruit the scaffolding protein eIF4G (EIF4G1, EIF4G2, or EIF4G3), which prevents the assembly of the eIF4F complex at the cap. This fundamental difference allows EIF4E2 to compete with eIF4E and block translation initiation of target mRNAs. The binding affinity of EIF4E2 for the cap is approximately 100-fold lower than eIF4E, and its cellular abundance is roughly 10-fold lower .

What types of EIF4E2 antibodies are available for research applications?

Current research employs both monoclonal and polyclonal antibodies against EIF4E2. Mouse monoclonal antibodies (such as clone 225CT3.1.3, IgG1,κ isotype) are available for western blotting and ELISA applications, typically generated against recombinant EIF4E2 protein. These recognize human EIF4E2 with high specificity . Rabbit polyclonal antibodies targeting epitopes within the N-terminal region (aa 1-150) of human EIF4E2 are suitable for immunocytochemistry and immunofluorescence applications . When selecting an antibody, researchers should consider the specific application needs and confirm species reactivity, as most commercially available antibodies are validated primarily for human samples .

What are the key cellular locations and functions of EIF4E2 that influence antibody selection?

EIF4E2 primarily localizes to the cytoplasm and more specifically to cytoplasmic processing bodies (P-bodies), where it functions in miRNA-mediated translational repression complexes. When designing immunofluorescence experiments, researchers should select antibodies validated for detecting both diffuse cytoplasmic signals and punctate P-body patterns. Additionally, EIF4E2 functions in association with GIGYF2 in the 4EHP-GYF2 complex to assist ribosome-associated quality control by sequestering the mRNA cap and blocking ribosome initiation on problematic messages. Therefore, antibodies that do not interfere with these protein-protein interactions are preferred for co-immunoprecipitation studies investigating EIF4E2's functional complexes .

How should researchers optimize western blotting protocols for EIF4E2 detection?

For optimal western blot detection of EIF4E2 (calculated molecular weight of 28.36 kDa), researchers should:

• Use RIPA or NP-40 lysis buffers containing protease inhibitors for sample preparation

• Load 20-40 μg of total protein per lane

• Employ reducing conditions with 10-12% SDS-PAGE gels for optimal separation

• Transfer to PVDF membranes (preferred over nitrocellulose for this protein)

• Block with 5% non-fat milk in TBST (BSA may be used for phospho-specific antibodies)

• Dilute primary monoclonal antibodies at 1:500-1:1000 as recommended by manufacturers

• Incubate overnight at 4°C for optimal signal-to-noise ratio

• Use appropriate HRP-conjugated secondary antibodies at 1:5000-1:10000 dilution

For subcellular fractionation studies, special attention should be paid to preserving cytoplasmic P-body structures where EIF4E2 concentrates during translational repression .

What controls and validation steps are necessary when using EIF4E2 antibodies in neuronal studies?

When studying EIF4E2 in neuronal systems, proper validation requires:

• Knockout/knockdown controls: Utilize conditional knockout models (such as 4EHP-eKO mice with Eif4e2 deleted in excitatory forebrain neurons) or siRNA knockdown in cultured neurons to confirm antibody specificity

• Developmental expression profiling: As EIF4E2 expression increases during development, age-matched controls are critical

• Brain region specificity: Validate antibody performance specifically in hippocampal tissue, where EIF4E2 regulates synaptic plasticity

• Subcellular localization confirmation: Confirm synaptosomal enrichment using synaptic markers co-staining

• Cross-reactivity assessment: Test for cross-reactivity with eIF4E and other family members using recombinant protein controls

• Species-specific validation: Separately validate antibodies for mouse and human samples, as epitope conservation may vary

These validation steps are particularly important when studying EIF4E2's role in synaptic plasticity mechanisms relevant to autism spectrum disorder models .

How can researchers effectively co-immunoprecipitate EIF4E2 with its binding partners?

To successfully co-immunoprecipitate EIF4E2 with interacting proteins such as GIGYF2 or TNRC6A:

• Use mild lysis conditions (e.g., 20 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.5% NP-40, with protease and RNase inhibitors) to preserve protein-protein interactions

• Pre-clear lysates with protein A/G beads to reduce non-specific binding

• For endogenous pulldowns, use at least 1-2 mg of total protein due to the relatively low abundance of EIF4E2

• Consider crosslinking antibodies to beads to prevent antibody chain interference during detection

• Include RNase treatment controls to distinguish RNA-dependent interactions

• For detecting interactions with TNRC6A, account for technical difficulties from low endogenous abundance of EIF4E2, potential degradation of TNRC6A, and limitations of available antibodies

• Consider overexpressing tagged versions (e.g., Flag-tagged EIF4E2) as an alternative approach when studying interactions with endogenous partners

• Use appropriate negative controls, such as IgG controls and comparison with eIF4E1 immunoprecipitation

Studies have shown that immunoprecipitation of endogenous TNRC6A can co-precipitate Flag-tagged EIF4E2 but not eIF4E1, confirming specificity of interaction .

How does EIF4E2 function under hypoxic conditions in cancer research models?

EIF4E2 plays a critical role in hypoxic cancer microenvironments through the following mechanisms:

• Cancer cells switch from eIF4E to eIF4E2-dependent cap-dependent translation under hypoxic conditions

• This switch is essential for cancer cells to form masses larger than ~0.15 mm (the oxygen diffusion limit)

• EIF4E2-directed protein synthesis is required for cancer cells to:

  • Survive and proliferate in low oxygen conditions

  • Form hypoxic tumor cores in in vitro spheroid models

  • Establish detectable tumors in xenograft assays

Importantly, eIF4E-directed protein synthesis alone cannot sustain cellular adaptation to hypoxia. Research approaches investigating this phenomenon should include:

• Comparative analysis of EIF4E2 expression in normoxic versus hypoxic conditions

• Knockdown studies examining spheroid formation capabilities

• In vivo tumor formation assays with EIF4E2-depleted cells

• Analysis of hypoxia response protein synthesis through metabolic labeling

These approaches have demonstrated that phenotypic expression of the cancer genome requires translation by the EIF4E2-directed hypoxic protein synthesis machinery .

What is the mechanistic role of EIF4E2 in microRNA-mediated translational repression?

EIF4E2 serves as a critical effector in microRNA-mediated translational silencing through several mechanisms:

• It competes with eIF4E for binding to 4E-T (eIF4E-Transporter), which is important for miRNA-mediated silencing

• TNRC6A (also known as GW182, a core component of RISC) can directly recruit EIF4E2 to target mRNAs to repress translation

• Immunoprecipitation studies have shown that endogenous TNRC6A co-precipitates with Flag-tagged EIF4E2 but not with eIF4E1

• The presence of a cap structure on target mRNAs optimizes miRNA silencing, suggesting cap-binding proteins like EIF4E2 are involved

• Earlier hypotheses suggesting Argonaute proteins might bind directly to the cap were challenged by subsequent structural studies

When designing experiments to study this mechanism, researchers should consider:

• Using reporter systems with miRNA binding sites to quantify translational repression

• Employing EIF4E2 knockdown/knockout approaches to assess effects on miRNA silencing efficacy

• Analyzing protein-protein interactions between EIF4E2, TNRC6A, and 4E-T

• Including appropriate controls to distinguish EIF4E2-dependent effects from other mechanisms

These approaches have established that EIF4E2 is required for efficient miRNA-mediated translational repression .

How does EIF4E2 contribute to autism spectrum disorder (ASD) pathophysiology in mouse models?

EIF4E2 plays a significant role in synaptic plasticity and behaviors relevant to ASD through specific neural mechanisms:

• EIF4E2 is expressed in excitatory neurons and synaptosomes, with expression increasing during development

• Conditional knockout of Eif4e2 in excitatory forebrain neurons (4EHP-eKO) results in:

  • Exaggerated metabotropic glutamate receptor long-term depression (mGluR-LTD), a synaptic plasticity phenotype frequently observed in ASD models

  • Social behavior impairments without deficits in olfaction, anxiety, locomotion, or motor ability

  • No effect on repetitive behaviors or vocal communication

• Interestingly, these phenotypes occur without changes in hippocampal global protein synthesis rates, suggesting EIF4E2 regulates translation of specific mRNA targets

• Heterozygous deletion of either Gigyf2, Eif4e2, or both genes did not produce ASD-like behaviors, indicating a gene dosage effect

Research approaches to investigate these mechanisms should include:

• Electrophysiological recordings of mGluR-LTD in hippocampal slices

• Behavioral assays focusing on social interaction paradigms

• Analysis of synapse-specific protein synthesis using techniques like FUNCAT

• Investigation of specific mRNA targets regulated by the EIF4E2-GIGYF2 complex

These findings link EIF4E2 function to ASD-relevant synaptic mechanisms distinct from global translational regulation .

How can researchers distinguish between EIF4E2's translation repression function and other cap-binding proteins in experimental systems?

To specifically attribute observed translational repression effects to EIF4E2 rather than other cap-binding proteins:

• Mutational approaches: Use EIF4E2 mutants with impaired cap-binding ability (based on structural studies) as negative controls

• Comparison with eIF4E: Include parallel experiments with eIF4E knockdown/overexpression to distinguish their roles

• Tethering assays: Employ MS2-based or λN-BoxB tethering systems to artificially recruit EIF4E2 to reporter mRNAs, bypassing the need for cap-binding

• Cap-binding competition assays: Utilize m7GTP-Sepharose pulldowns with increasing concentrations of free cap analog to compare binding affinities

• Structure-informed antibody selection: Choose antibodies that do not interfere with the cap-binding pocket when studying EIF4E2's function

• GIGYF2 interaction: Assess the requirement for GIGYF2 binding, as this interaction is specific to EIF4E2-mediated repression

• Specific target mRNAs: Focus on mRNAs known to be regulated by EIF4E2 but not other cap-binding proteins

These approaches help delineate EIF4E2's specific contribution to observed translational regulation effects and avoid attributing phenotypes to the wrong cap-binding protein .

What are the critical technical considerations when studying EIF4E2's role in ribosome-associated quality control?

When investigating EIF4E2's function in ribosome-associated quality control (RQC), researchers should:

• Distinguish parallel pathways: Design experiments to differentiate between EIF4E2-GIGYF2 activity and RQC-mediated degradation of stalled nascent polypeptides

• Account for ZNF598 independence: Include controls to verify that EIF4E2-GIGYF2 works downstream and independently of ZNF598, while recognizing that ZNF598 may recruit them to faulty mRNAs

• Use appropriate stalling reporters: Employ reporter constructs containing ribosome stalling sequences (e.g., polyA sequences, rare codons)

• Measure both mRNA and protein levels: Assess effects on both mRNA stability and translation efficiency

• Employ ribosome profiling: Use techniques like ribosome profiling to precisely map ribosome stalling sites and assess EIF4E2's effect on ribosome distribution

• Consider RNA degradation pathways: Include controls for No-Go Decay and other RNA quality control mechanisms

• Account for potential redundancy: Design knockdown studies that address functional redundancy with other translational repressors

These considerations help isolate EIF4E2's specific contribution to quality control of problematic mRNAs from other parallel cellular mechanisms .

What approaches can resolve contradictory data regarding EIF4E2's role in different experimental systems?

When faced with contradictory findings about EIF4E2 function across different experimental systems, researchers should systematically address potential sources of variation:

• Cell type specificity: Compare neuronal (e.g., hippocampal excitatory neurons) versus non-neuronal systems, as EIF4E2 may have tissue-specific roles

• Developmental timing: Assess whether contradictions arise from studying different developmental stages, as EIF4E2 expression increases during development

• Oxygen conditions: Control for hypoxic versus normoxic conditions, as EIF4E2 has specialized functions under hypoxia in cancer cells

• Interaction partners: Verify expression levels of key partners like GIGYF2 across experimental systems

• Methodological differences: Standardize antibody selection, detection methods, and quantification approaches

• Target mRNA populations: Employ transcriptome-wide approaches (RIP-seq, CLIP-seq) to identify cell-type-specific EIF4E2 targets

• Post-translational modifications: Investigate whether EIF4E2 regulation via phosphorylation or other modifications differs between systems

• Genetic background effects: In mouse models, control for strain-specific effects by using appropriate genetic controls

A structured approach to reconciling contradictory data will advance understanding of context-specific EIF4E2 functions and avoid overgeneralizing findings from a single experimental system .

What emerging technologies can advance the study of EIF4E2's translation regulation mechanisms?

Several cutting-edge technologies show promise for elucidating EIF4E2's precise molecular mechanisms:

• Proximity labeling: BioID or TurboID fusions with EIF4E2 can identify transient interaction partners in living cells with spatial and temporal resolution

• Single-molecule imaging: Techniques like single-molecule fluorescence in situ hybridization (smFISH) combined with immunofluorescence can visualize EIF4E2-targeted mRNAs in real-time

• Cryo-electron microscopy: Structural studies of EIF4E2-containing complexes can reveal conformational changes during translational repression

• Ribosome profiling: Specialized ribosome profiling approaches focusing on initiating ribosomes can quantify EIF4E2's effect on translation initiation with nucleotide resolution

• CRISPR-based screens: Genome-wide CRISPR screens can identify novel genetic interactions with EIF4E2 in various cellular contexts

• Translatomics: Techniques combining polysome profiling with RNA sequencing can identify the complete set of mRNAs regulated by EIF4E2 under different conditions

• Patient-derived models: iPSC-derived neurons from patients with GIGYF2 mutations can provide physiologically relevant models for studying EIF4E2 function in disease

These advanced approaches will help resolve current knowledge gaps regarding EIF4E2's selective targeting of specific mRNAs and its context-dependent functions .