Phospho-S6 Detection Kit

Phospho-S6 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-S6 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. Stained phospho-S6 in A549 cells treated with media (non-treated) or with the mTOR inhibitor rapamycin (50 nM) for 16 hours. Cells were fixed and stained according to the kit protocol with Hoechst 33342 dye for nuclei (blue) and Phospho-S6 primary antibody plus DyLight 549 Goat Anti-Rabbit Secondary Antibody (orange). Stained cells then were imaged using the ArrayScan® VTI HCS Reader. Treatment with Rapamycin inhibits expression of phospho-S6.

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 leading its substrate S6 phosphorylation is important in regulating protein synthesis, cell size, cell-cycle progression, glucose homeostasis and survival of newborns. The translation efficiency of mRNAs with a 5´-terminal oligopyrimidine tract (‘TOP mRNAs’) correlates under some physiological circumstances with levels of S6K activation and its substrate S6 phosphorylation. The phosphorylation sites in S6 have been mapped to five clustered residues, Ser235, Ser236, Ser240, Ser244 and Ser247, whose location at the C terminus of higher eukaryotes is evolutionarily conserved.

This kit was optimized using the Cellomics 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-S6 expression. In addition to HCS instruments, cells stained using reagents in this kit can be viewed and analyzed by fluorescence and confocal microscopes.

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.  

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.