Figure 1.

ULK1 induces phosphorylation of Raptor. (A) HEK293 cells transfected with Myc-mTOR, HA-Raptor, HA-mLST8 in addition to V5-tagged ULK1 (where indicated), were radiolabeled with [³²P]-orthophosphate. After lysis, mTOR/Raptor/mLST8 was immunoprecipitated using anti-HA antibodies and phosphorylation of mTORC1 components assessed by autoradiography. (B) HA-Raptor and V5-ULK1 were co-expressed in HEK293 cells and Raptor was purified by HA-immunoprecipitation. One sample was treated with SAP, as indicated. Western blots were performed to determine the mobility of Raptor. (C) HEK293 cells were co-transfected with HA-Raptor and either wild-type (WT) or kinase dead (KD) V5-ULK1. Following HA-immunoprecipitation, samples were assessed for Raptor mobility by western blot. (D) As in (C) but cells were radiolabeled with [³²P]-orthophosphate prior to lysis and immunoprecipitation. Phosphorylation of Raptor was determined by autoradiography.