a, Interaction between purified recombinant ATG14 and GST–Flag-tagged (GF) autophagic SNAREs using an in vitro GST pull-down assay followed by western blot (IB, immunoblot). b, Recombinant ATG14 binds to STX17–SNAP29 binary t-SNARE complex but not STX17–SNAP29–VAMP8 ternary complex using an in vitro immunoglobulin-G (IgG) pull-down assay followed by western blot. c, Flag–STX17, GFP–ATG14, endogenous LC3, LAMP2 and ATG16 were detected by immunostaining in EBSS-starved U2OS cells (n =20). Scale bars, 5 μm. d, Corresponding statistical analysis of co-localization of ATG14, STX17 and LC3 upon treatment with EBSS or chloroquine (CQ).