Figure 3.

ULK1 phosphorylates ATG13 and Raptor in vitro. (A) An in vitro ULK1 kinase assay was performed by immunoprecipitation of either wild-type (WT) or kinase dead (KD) V5-ULK1 followed by incubation with substrate (either purified GST-ATG13 or GST-Raptor) for 45 min in the presence of γ-[³²P]-ATP. [³²P]-radiolabel incorporation into substrates was determined by autoradiography. (B) The assay was performed as in (A) using GST-Raptor as substrate and cold ATP. Samples were assessed for Raptor phosphorylation using site-specific phospho-Raptor antibodies. Total Raptor and ULK1 are shown as controls.