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mTORC1 activity negatively regulates the CDK8 and CycC protein levels.

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posted on 2015-06-04, 03:25 authored by Daorong Feng, Dou Yeon Youn, Xiaoping Zhao, Yanguang Gao, William J. Quinn 3rd, Alus M. Xiaoli, Yan Sun, Morris J. Birnbaum, Jeffrey E. Pessin, Fajun Yang

A) Effects of mTOR knockdown on the levels of indicated proteins during nutrient-depletion or nutrient-repletion. HEK293T cells were infected with non-specific (NS) or mTOR-specific shRNA lentiviruses. The cells were nutrient-depleted by placing them in the low serum DMEM medium overnight, and then in amino acid-free RPMI for 2 hour followed by the addition of regular DMEM containing 100 nM insulin for 30 min. The relative protein levels of CDK8 (B) and CycC (C) were further analyzed by densitometry. *p<0.05 vs. control (n = 3). D) Effects of overexpressing a mutant of constitutively active Rheb or Ulk1 on the levels of indicated proteins during nutrient-depletion (ND) or nutrient-repletion (NR) condition. HEK293T cells were transfected with a constitutively active ULK1 mutant cDNA (U, 1 μg), the empty vector (V, 1 μg) or a constitutively active Rheb mutant cDNA (R, 1 μg). Thirty-six hours after transfection, the cells were nutrient-depleted and nutrient-replete as described above. These are representative immunoblots independently performed for five times.