mTORC1 activity negatively regulates the CDK8 and CycC protein levels.

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.