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The Development And Molecular Expression In Mammalian Cells Of An Ha-Tagged Plasmid Encoding For The Target Of Rapamycin (Mtor), Kevin S. Dougherty
The Development And Molecular Expression In Mammalian Cells Of An Ha-Tagged Plasmid Encoding For The Target Of Rapamycin (Mtor), Kevin S. Dougherty
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The protein kinase mammalian Target of Rapamycin (mTOR) is a master controller of cell growth and proliferation due to its ability to integrate growth signals and regulate translation through 4E-BP1 and p70-S6K. We developed four phCMV2-HA-tagged plasmids to overexpress TOR in mammalian cells: Wild Type, D2357E (Kinase Dead), R2109A (PA Binding Deficient) and, S2035T (Rapamycin Resistant) each verified by restriction enzyme digestion and direct sequencing. Plasmid DNA was overexpressed in COS-7 cells and the 289 kDa protein detected in Western blots developed with anti-HA and anti-mTOR antibodies. Kinase activity of the overexpressed protein was detected by in vitro phosphorylation of …