Eukaryotic initiation factor 4E binding protein 1 (4E-BP1), also known as PHAS, is a ~20 kDa member of a family of eIF4E-binding proteins whose binding affinity to eIF4E is regulated by its phosphorylation. It inhibits cap-dependent translation by binding to eIF4E on the same site that overlaps the binding site for eIF4G, preventing its binding to the latter and eventually leading to an increase in mRNA translation. The phosphorylatin of 4E-BP1 is critical in determining cell fate by controlling translation initiation and apoptotic potency. 4E-BP1 is hyperphosphorylated in response to several external stimuli including hormones, growth factors, mitogens, cytokines and G-protein–coupled receptors and in response to stress conditions including nutrient deprivation. The phosphorylation of 4E-BP1 increases in response to activated phosphoinositol 3'-kinase (PI-3K) or its downstream effector Akt/PKB. 4E-BP1 is believed to mediate PI-3K and FRAP/mTOR signaling and is phosphorylated on at least six serine and threonine sites (Thr 37, Thr 46, Ser 65, Thr 70, Ser 83, and Ser 112). The phosphorylation of these sites is believed to occur in an orderly fashion where phosphorylation of threonine 37 and 46 by FRAP/mTOR is a priming step for subsequent phosphorylation of 4E-BP1 at the carboxy-terminal sites.
Chemically synthesized phosphopeptide derived from the region of human 4E-BP1 that contains threonine 46. The sequence is conserved in human, mouse and rat.
NIH3T3 cell lysate
Provided as solution in phosphate buffered saline, pH 7.3, with 1.0 mg/ml BSA and 0.05% sodium azide
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Lysates prepared from NIH3T3 cells left untreated or treated with PI-3K inhibitor LY2904002 (lanes 2 & 3), prior to PDGF stimulation (lanes 1 & 3) and from EGF-treated HEK293 cells (4-8), Membranes were then left untreated (lanes 1-7) or treated with Lambda phosphatase (8), and incubated with 4E-BP1 [pT46] antibody for two hours at room temperature, following prior incubation with: no peptide (lanes 1 & 2), non-phosphopeptide corresponding to the immunogen (3), a generic phospho-threonine-containing peptide (4), or the phosphopeptide immunogen (5).
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