Fig. 3

ATG14 phosphorylation promotes Vps34 lipid kinase activity. a Vps34 lipid kinase assay. Empty vector or FLAG-ATG14 S29 WT, A, or E mutants were overexpressed in HEK cells and pulled down using antibodies against FLAG. b Quantification of PI(3)P levels were normalized to levels of Vps34 in the IP. Values were then normalized to the WT condition. **p < 0.01 (n = 5). Data are represented as mean +/− SEM. c Co-immunoprecipitation of ATG14 and Vps34 complex members, Beclin 1, Vps34, Vps15 and NRBF2. Empty vector or FLAG-ATG14 S29 WT, A, or E mutants were overexpressed in HEK cells and pulled down using antibodies against FLAG. d Torin 1 treatment does not alter binding of Vps34 complex members. MEF cells were treated with Torin 1 for 60 min. Cell lysates were subject to IP using antibody against ATG14. e Phosphorylation levels of Beclin 1 and ATG14 from HCT116 cells treated with Torin 1 for 15, 30, 60, and 120 min. f Vps34 lipid kinase assay for Beclin 1 and ATG14 phosphorylation mutation comparison. Beclin 1-AsRed, FLAG-ATG14, and dual myc-Vps34 his-Vps15 plasmid were overexpressed and purified from HEK cells using antibodies against FLAG. g Quantification of PI(3)P levels were normalized to levels of Vps34 in the IP. Values were then normalized to the WT condition. Two-way ANOVA analysis with Bonferroni posttest was used for the effect of the nature of the mutation and which protein was being mutated. Main effect of Beclin 1 or ATG14 mutation F(1, 18) = 4.08, p = 0.0587; Main effect of type of mutation F(1, 18) = 0.71, p = 0.5043; Interaction F(2, 18) = 3.80, p = 0.0421. **p < 0.01 (n = 5). Data are represented as mean +/− SEM