Phospho-4E-BP1 Detection Kit

Phospho-4E-BP1 is measured directly using a fixed end-point assay based on immunofluorescence detection in cells grown on standard high-density microplates. This kit contains an anti-phospho-4E-BP1 primary antibody (rabbit monoclonal), a goat anti-rabbit secondary antibody conjugated to DyLight™ 549 Fluorophore and various other reagents and buffers required for immunofluorescence staining for high-content screening (HCS) assays.

Figure 2. Staining of phospho-4E-BP1 in HeLa cells treated with media (non-treated) or with Ly294002 (100 µM) for 16 hours. Cells were fixed and stained according to the kit protocol with Hoechst 33342 dye for nuclei (blue) and Phospho-4E-BP1 primary antibody plus DyLight 549 Goat Anti-Rabbit Secondary Antibody (orange). Stained cells then were imaged using the ArrayScan V^TI HCS Reader. Treatment with Ly294002 inhibits expression of phospho-4E-BP1.

Downstream effectors of mTOR-induced translational control include ribosomal protein S6 kinase (S6K) and the eukaryotic initiation factor 4E (eIF4E)-binding protein (4E-BP1). The 4E-BP1 (eIF4E-binding protein 1, also called PHAS-I) is a small heat- and acid-stable phosphoprotein whose phosphorylation is enhanced by growth factors, serum stimulation and insulin in a PI3 kinase-dependent manner. 4E-BP1 undergoes phosphorylation at several sites leading to its release from eIF4E and allowing eIF4E to bind eIF4G and form initiation complexes that facilitate upregulation of protein synthesis.

Figure 3. Downstream effectors of mTOR induced translational control include ribosomal protein S6 kinase (S6K) and the eukaryotic initiation factor 4E (eIF4E)-binding protein (4E-BP1). Activation of S6K and its substrate S6 phosphorylation is blocked by mTOR inhibitor rapamycin. Phosphorylation of 4E-BP1 is inhibited by the PI3 kinase inhibitor Ly294002.

This kit was optimized using the Thermo Scientific ArrayScan® HCS Reader and the Compartmental Analysis BioApplication Software Module but can be used with other Cellomics BioApplications. Thus, automated plate-handling, focusing, cell image acquisition and data analysis are combined in one HCS system to assay for compounds that regulate phospho-4E-BP1 induction. In addition to HCS instruments, cells stained using reagents in this kit can be viewed and analyzed by fluorescence and confocal microscopes.

       

Figure 4. Dose response curves for phospho-S6 in A549 and HeLa cells. The average intensities of phospho-S6 in the cytoplasm are plotted in response to rapamycin or Ly294002 concentration in cells treated for 16 hours. EC₅₀ values for both inhibitors and cell types are listed.

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