Figure 6
From: HIF-independent role of prolyl hydroxylases in the cellular response to amino acids
PHDs signal to mTORC1 in a HIF/TSC-independent manner. (a) U2OS cells were transfected with a plasmid encoding control, nontargeting shRNA (shCntr) or transfected with a plasmid encoding shRNA for HIF1b (clone no. 4; shHIF1b no. 4). After 48 h, cells were starved for amino acids for 2 h and then incubated for 20 min with (+ AA) or without (− AA) amino acids either in the presence or absence of DMOG. Accumulation of REDD1 and phosphorylation state of S6K and 4EBP1 were analyzed by western blot. (b) Phosphorylation status of S6K in TSC2+/+ and TSC2−/− mouse embryonic fibroblasts starved for 2 h and then re-fed with amino acids for one more hour in the presence or absence of DMOG. (c) U2OS cells were transfected with either empty vector or Flag-Rheb-GTP, as indicated. After 48 h, cells were amino-acid starved for 2 h and re-stimulated for 1 h with amino acids (AA) either in the presence or absence of DMOG, as indicated. Phosphorylation of S6K and S6 was then measured by immunoblotting. (d) U2OS cells were transfected with either empty vector or with HA-RagB-GTP plus HA-RagC-GDP, as indicated. After 48 h, cells were amino-acid starved for 1 h where indicated, DMOG was added 30 min before the collection of the cells. Phosphorylation of S6K and S6 was then measured by immunoblotting.
