Fig. 2

ATG14 is phosphorylated in an mTOR-dependent fashion by ULK1 upon autophagy induction. a Phosphorylation of ATG14 in HCT116 cells after glucose, serum, or amino acid withdrawal, medium starvation with HBSS, or Torin 1 treatment for 6 h. b ATG14 phosphorylation levels were normalized to total ATG14 levels and compared to the control condition. **p < 0.01 *p < 0.05 n.s. not significant (n = 3). Data are represented as mean +/− SEM. c ATG14 phosphorylation after 15, 30, 60, and 120 min of Torin 1 treatment in HCT116 cells. d ATG14 phosphorylation levels were normalized to total ATG14 levels and compared to the control condition. A one-way ANOVA with Bonferroni’s posttest was performed. F(4, 10) = 12.58, p = 0.0006 ***p < 0.001 **p < 0.01 *p < 0.05 (n = 3). Data are represented as mean +/− SEM. e ATG14 phosphorylation in Beclin WT, KO or Beclin rescued MEF cells treated with Torin 1 or vehicle (DMSO) for 60 min. Arrow indicates pATG14 band. f Quantification of ATG14 phosphorylation normalized to total ATG14. **p < 0.01 (n = 3). Data are represented as mean +/− SEM. g MEF WT and ULK1/2 DKO cells were treated with Torin 1 for 60 min. ATG14 was immunopurified to enhance the phospho-signal. h Validation of phospho-ATG14 (S29) antibody specificity. ATG14 phosphorylation signal disappears in ATG14 KO HCT116 cells after medium starvation or Torin 1 treatment for 60 min