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. 2011 Oct 1;7(10):1212–1221. doi: 10.4161/auto.7.10.16660

Figure 1.

Figure 1

ULK1, ULK2 and Atg13, but not Atg5, negatively regulate mTORC1 signaling. (A) Knockdown of ULK1, ULK2 and Atg13, but not Atg5, increased mTORC1 signaling. 293T cells were transduced by Atg13, ULK1, ULK2 or scrambled shRNA (control). The phosphorylation of s6K1 (p-Thr389) and the expression levels of proteins were analyzed by protein gel blotting. β-actin was a loading control. (B) Overexpression of ULK1 inhibited S6K1 phosphorylation. Cell lysate was obtained from 293T cells overexpressing myc-tagged ULK1 or cells transduced by mock vector. The phosphorylation state of endogenous S6K1 (p-Thr389) was analyzed by protein gel blotting. (C) ULK1 deficiency increased leucine-stimulated S6K1 phosphorylation. ULK1 MEFs were cultured in RPMI medium deprived of leucine for 40 min before cells were supplemented with leucine (52 µg/ml) for different periods of time. The phosphorylation state (p-Thr389) and expression level of S6K1 were analyzed by protein gel blotting. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was a loading control. (D) ULK1 deficiency increased insulin-stimulated S6K1 phosphorylation. ULK1 MEFs were incubated in DMEM without serum overnight and treated with insulin (10 nM) for different periods of incubation time. The phosphorylation states of S6K1 (p-Thr389) and Akt (p-Ser473) and their expression levels were analyzed by protein gel blotting. β-actin was monitored as a loading control. (E) ULK1 knockdown increasesd S6K1 phosphorylation in 293T cells. 293T cells transduced by ULK1 shRNA or scrambled shRNA (control) were treated with insulin (100 nM) as described in (D). The phosphorylation states and expression levels of proteins were analyzed by protein gel blotting. (F) ULK2 knockdown increased S6K1 phosphorylation in 293T cells. The cells transduced by ULK2 shRNA or scrambled shRNA (control) were treated with insulin, and the phosphorylation state of S6K1 was analyzed as described (D). (G) Atg5 deficiency did not increase S6K1 phosphorylation. Atg5 MEFs were treated with insulin as described in (D).