Fig.4. ATG14L stimulates Beclin-1 S14 phosphorylation by promoting association with ULK1.

Unless otherwise stated all experiments were repeated three times and data shown is representative. (a) Beclin-1 alone (lanes 1-4) or Beclin-1 and ATG14L (lanes 5-8) were overexpressed in 293 cells. Beclin-1 was purified either by direct immunoprecipitation (lanes 1-4) or by ATG14L IP (lanes 5-8). IP samples were subjected to an in vitro ULK1 kinase assay with increasing amounts of ULK1. Reactions were immunoblotted with the indicated antibodies. Black line denotes discontinuous lanes from the same gel. Two unique experiments were performed. (b) Beclin-1 alone or bound to ATG14L was purified as described in panel a. Equal amounts of ULK1 were added to each complex and reactions were quenched at the indicated time points. Western blot was performed with the indicated antibodies. (c) An ATG14L-FLAG-6His inducible U2OS cell line was induced for 16 hours in the presence of amino acids. Endogenous Beclin-1 was immunoprecipitated and immunoblotted as in Fig.3a. ATG14L input levels are detected by immunoblotting. Two unique experiments were performed. (d) 293 cells transfected with either ATG14L or Beclin-1, or both, in conjunction with ULK1 were immunoprecipitated as indicated and blotted with the indicated antibodies. (e) 293 cells were transfected with Beclin-1 and ULK1 in the presence of ATG14L-WT or ATG14LΔCCD, which is defective in Beclin-1 binding, under nutrient rich conditions. Lysates were resolved by SDS-PAGE and blotted with the indicated antibodies. (f) 293 cells were transfected with ULK1 and Beclin-1 in conjunction with either ATG14L-WT, or one of two mutants (ΔBATS, ΔN) that are defective in phagophore localization. Samples were handled as in panel e.