Fig. 4

mTORC1 stabilizes Skp2 via ubiquitin/proteasome pathway. a 293 T cells transfected with Control or mTOR-siRNA were incubated with 20 μg/ml of CHX for the indicated times. Cell lysates were then analyzed by Western blot (left panel), and the densities of the Skp2 protein bands at each time points were normalized to GAPDH (right panel), indicating Skp2 degrades much faster in mTOR-depleted cells than control cells. b 293 T cells stably expressing Skp2-WT or the S64A mutant were incubated with 20 μg/ml of CHX for the indicated times. Cell lysates were then analyzed, indicating Skp2 displayed decreased half-life in cells expressing Skp2-S64A compared to those expressing Skp2-WT (c, d) 293 T cells were transfected with the indicated plasmids, followed by immunoprecipitation with nickel-agarose beads and then analyzed by Western blot. The ubiquitinated Skp2 decreased in mTOR overexpression cells, indicating that the degradation of Skp2 prevented by mTORC1 is ubiquitination dependent (c). In the presence of Skp2-S64A, Skp2 was degrades faster than Skp2-WT (d)