The kit contains a monoclonal rabbit anti-phospho-mTOR antibody, a goat anti-rabbit secondary antibody conjugated to Thermo Scientific DyLight 549 Fluorophore and various other reagents and buffers required for immunofluorescence detection of mTOR for high-content screening (HCS) assays.
The mammalian target of rapamycin (mTOR) is a protein kinase that regulates the initiation of protein translation, significantly influencing cell growth. Consequently, mTOR is responsive to a diversity of cellular signals including growth factors, nutrients and the cellular metabolic state. Two of the major signaling pathways that regulate mTOR activation are the PI-3 kinase through RTK signal transduction and AMPK, an energy sensing pathway. Under favorable growth conditions, the negative regulators of mTOR, TSC1 and 2 are phosphorylated by AKT, allowing mTOR to dissociate from the TSC complex for activation. mTOR is then phosphorylated, and through phosphorylation activates S6 kinase and dissociates 4E-BP-1 from the cap complex to promote protein translation. Cancer cells are often mutated in the PI-3 kinase and mTOR pathways to promote constitutive cell growth, and members of this pathway are attractive targets for anti-cancer drugs.
Phospho-mTOR was activated after a brief stimulation with serum and insulin in serum-starved or rapamycin-treated C2C12 muscle cells resulting in an increase in cytosolic intensity, visualized as spots in the perinuclear region of the cell. The phospho-mTOR assay was optimized with the Thermo Scientific ArrayScan Reader using the Compartmental Analysis BioApplication Software Module. Phospho-mTOR can be quantitated using the cytoslic intensity or the cytosolic spot intensity. Cells stained using this kit also can be imaged using fluorescence or confocal microscopy.
Figure 1. Activation of phospho-mTOR after serum stimulation in C2C12 muscle cells. Cells were serum-starved for 24 hours, followed by 15 minute stimulation with 10% serum and 50 ng/ml insulin. Cells were then fixed and stained according to the kit protocol. Cells were labeled with Hoechst 33342 dye for nuclear staining and imaged using the ArrayScan VTI HCS Reader.
Figure 2. Dose-response curves of phospho-mTOR in cells treated with serum after serum-starvation. C2C12 cells were treated for 15 minutes with serum and insulin. EC50 = 0.37 ± 0.27% serum and 0.037 ± 0.027 µg/ml insulin. HeLa cells were treated for 15 minutes with serum. IMR-90 cells were treated for 15 minutes with serum. EC50 = 0.1% serum. Each data point represents eight wells. C2C12 curves represent separate data from three replicate plates. Single curves represent data from a single plate. Serum concentrations were derived from serial dilutions of five- or ten-fold, where 2 = 100%, and 1 = 10%.
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