Arfaptins are associated with ATG9A vesicles. (A) Overview of the immunoisolation procedure used to purify ATG9A-positive membranes. Ab14F2 is a hamster monoclonal antibody raised against a C-terminal epitope (Webber and Tooze, 2010). (B) Scatterplot of proteins associated with ATG9A-positive membranes from cells incubated in FM or EBSS for amino acid depletion (ES). The x axis displays the Log2 of the SILAC ratio (ES/FM), and the y axis displays the Log10 of the intensity. ATG9A and examples of proteins enriched or depleted in ATG9A-positive membrane upon autophagy induction are highlighted. See Table S1. (C) Selected GO categories illustrate proteins associated with ATG9A-positive membranes from cells treated as in B. (D) HEK293A cells were incubated in FM or ES for 2 h, and the ATG9A-positive compartment was immunoisolated before detection of ATG9A, ARFIP1, and ARFIP2 by immunoblot. (E) Quantification of the relative amount of ARFIP1 and ARFIP1 after normalization to immunoisolated ATG9A in D. Statistical analysis using two-tailed unpaired Student’s t test, mean ± SEM, n = 3 experiments; *, P ≤ 0.05; **, P ≤ 0.01. (F) HEK293A cells were incubated in FM or ES for 2 h, fixed, and labeled using antibodies to ARFIP2 and ATG9A. Airyscan imaging. Scale bars, 5 µm; inset, 1 µm. Line scans of ARFIP2 and ATG9A labeling in FM (G) or ES (H) from F.