Fig. 1

ULK1 phosphorylates ATG14 at serine 29. a Representation of ATG14 structure with the location of serine 29. 4C-ER – cysteine repeats needed for ER localization. CCD – coiled-coil domains. BATS – BARKOR autophagosome targeting sequence. b Co-immunoprecipitation of ATG14 and ULK1. Myc-ULK1 and ATG14-GFP were overexpressed in HEK cells and pulled down using antibodies against GFP. c Autoradiogram from in-vitro kinase assay with ULK1 and ATG14 a.a. 20–73. Myc-ULK1 WT, KI, or empty vector was overexpressed in HEK cells and immunopurified with antibodies against myc. ATG14 a.a. 20–73 WT, S29A, or S61A were purified from e. coli. MBP (myelin basic protein) was used as a positive control. d Similar experiment as (c), except using an antibody against phosphorylated serine 29. e ATG14-GFP and myc-ULK1 WT or KI were co-transfected in HEK cells. Half of the IP was treated with an alkaline phosphatase at 37 °C for 30 min