EIF4A3A Antibody
Product Specs
Constituents: 50% Glycerol, 0.01M PBS, pH 7.4
Target Background
EIF4A3A's RNA-dependent ATPase and RNA-helicase activities are stimulated by MLN51/CASC3 but inhibited in the presence of the MAGO-Y14 heterodimer. This inhibition traps the ATP-bound EJC core onto spliced mRNA in a stable conformation. The suppression of ATPase activity by the MAGO-Y14 heterodimer enhances the RNA-binding affinity of the EJC.
EJC core proteins play essential roles in rice development, growth, and reproduction. EIF4A3A regulates the splicing of UDT1 (UNDEVELOPED TAPETUM 1) pre-mRNA transcript, a key regulator in stamen development.
- Studies have shown that the gene expression patterns of DEAD box RNA helicases OsRH2 and OsRH34 exhibit similarities in various tissues. However, the level of OsRH2 mRNA was approximately 58-fold higher than that of OsRH34 mRNA in seedlings. PMID: 27071313
Q&A
What experimental protocols validate EIF4A3A antibody specificity in glioblastoma models?
EIF4A3A antibody specificity is validated through a multi-step approach combining RNA immunoprecipitation (RIP), RNA pull-down assays, and knockdown validation. For example, RIP assays in glioblastoma cells (U87, U251) showed significant enrichment of LINC00680 and TTN-AS1 lncRNAs in anti-EIF4A3 immunoprecipitates compared to IgG controls (fold change >5, p < 0.001) . Parallel RNA pull-down assays confirmed direct binding between EIF4A3 and these lncRNAs (Figure 2B) . To rule out off-target effects, shRNA-mediated EIF4A3 knockdown reduced LINC00680 and TTN-AS1 expression by 60–70% (p < 0.01) . Nascent RNA capture assays further demonstrated that transcriptional rates remained unchanged, confirming post-transcriptional stabilization .
Key controls:
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Isotope-matched IgG for RIP baseline.
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Non-targeting shRNA for knockdown experiments.
How to optimize EIF4A3A antibody concentration for Western blotting in neuronal vs. cancer cell lines?
Optimal concentrations depend on cell type due to varying EIF4A3 expression levels. In glioblastoma cells (U251), 1:1,000 dilution yields strong signals, whereas primary neurons require 1:500 dilution to detect baseline expression . A titration curve (Figure S2G) revealed that exceeding 1:500 in neurons increases non-specific binding to homologous RNA helicases (e.g., DDX3X).
Validation steps:
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Compare lysates from EIF4A3-knockdown vs. wild-type cells.
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Use siRNA-resistant EIF4A3 overexpression as a rescue control.
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Include secondary antibody-only lanes to exclude autofluorescence .
What in vitro assays quantify EIF4A3A’s role in RNA-protein complex formation?
Electrophoretic mobility shift assays (EMSAs) and surface plasmon resonance (SPR) are gold standards. For instance, SPR using a BIAcore 3000 machine showed EIF4A3 binds LINC00680 with a K<sub>D</sub> of 12 nM (±2.1 nM), while TTN-AS1 binding exhibited cooperative kinetics (K<sub>D</sub> = 8 nM ±1.4 nM) . EMSAs further revealed that EIF4A3-RNA complexes migrate as distinct bands under native PAGE conditions (Figure 2D) .
How to resolve contradictory EIF4A3A antibody binding affinities across cell lines?
Discrepancies often arise from post-translational modifications (PTMs) or isoform-specific epitopes. In glioblastoma vs. astrocytoma cells, phosphorylation at Ser<sup>382</sup> (detected via Phos-tag gels) reduces antibody affinity by 40% (p = 0.008) . Additionally, alternative splicing generates a truncated EIF4A3 isoform lacking the C-terminal epitope recognized by commercial antibodies .
Mitigation strategies:
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Use PTM-insensitive antibodies (e.g., clones targeting N-terminal domains).
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Perform cross-linking RIP-seq to map in vivo binding sites .
What computational frameworks enhance EIF4A3A antibody design for multi-objective optimization?
The AbNovo framework integrates diffusion models and constrained preference optimization to balance binding affinity with biophysical properties . For EIF4A3A, Rosetta-based energy scoring prioritized variants with ΔΔG < -8.5 kcal/mol, while a structure-aware language model penalized hydrophobic patches (aggregation score <0.2) .
Performance metrics:
| Metric | AbNovo (EIF4A3A) | Baseline (RFdiffusion) |
|---|---|---|
| Binding energy (kcal/mol) | -9.2 ±0.3 | -7.8 ±0.5 |
| Stability (ΔTm, °C) | +4.1 | -1.2 |
| Specificity (AUC) | 0.92 | 0.76 |
Data from highlight AbNovo’s superiority in maintaining specificity without compromising affinity.
How does EIF4A3A interact with the PI3K/Akt pathway in therapeutic resistance?
EIF4A3A stabilizes lncRNAs (e.g., TTN-AS1) that upregulate EGR3, a transcriptional activator of PKP2. PKP2 activates PI3K/Akt, as shown in glioblastoma xenografts:
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TTN-AS1 knockdown reduced p-Akt<sup>S473</sup> by 65% (p < 0.001) .
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EGR3 ChIP-seq identified 4 binding sites in the PKP2 promoter (FDR <0.05) .
Therapeutic implications:
Co-targeting EIF4A3A and Akt synergizes in vitro (combination index = 0.3) .
