Fig. 2

ATG4B Ser316 is a target residue of ULK1 phosphorylation. a Clustal Omega multiple alignment of ATG4 protein sequences surrounding hATG4B serine 316 from different isoforms and species; highlighted in green are serine residues and in violet hydrophobic amino acids. b In vitro radioactive phosphorylation assay with WT or ATG4B S316A mutant at different time points after addition of recombinant active ULK1. Coomassie blue staining is shown as an ATG4B loading control. c GST-ATG4B or GST-ATG4B S316A mutant was incubated for the indicated times with GST-ULK1 (1–283); samples were then assayed with a custom phospho-specific antibody against Ser316 of human ATG4B—pATG4B (Ser316) or total ATG4B. d Total lysates from wild-type (WT) or ULK1/2 double knockout (DKO) mouse embryonic fibroblasts (MEFs) transfected with 3×-FLAG-tagged ATG4B or mCherry as a control and treated with 1 µM okadaic acid for 1 h were probed with the pATG4B(Ser316) antibody and with an anti-ACTIN antibody as loading control. In parallel, lysates from the same samples were subjected to immunoprecipitation with FLAG M2 affinity gel and probed for pATG4B(Ser316) and FLAG antibody. Phosphorylation level was calculated using densitometry, with pATG4B(Ser316) signal divided by FLAG signal for the same band, expressed as a percentage of the WT MEF IP sample. e HEK293T lysates from samples co-transfected with mouse WT myc-ULK1 or Kinase Inactive (KI) myc-ULK1 and the indicated Halo constructs for 24 h were blotted and probed with the indicated antibodies (one representative blot from three independent experiments)