(A and B) In vivo depletion of ERGIC abolishes the in vitro lipidation of LC3. Atg5 KO MEFs were treated without or with 10 µg/ml Brefeldin A (BFA) for 30 min and then incubated with the indicated concentrations of H89 for 10 min (A). Alternatively, cells were directly treated with the indicated drugs: 100 µM H89, 500 µM clofibrate, 50 µM kbNB142-70 and 50 µM Pitstop2 (B). Total membranes from the cells were collected, the lipidation reaction with T7-LC3 was performed and the products evaluated by SDS-PAGE and immunoblot. Ctr, control (C) Blocking ERGIC disruption preserved the in vitro lipidation of LC3. Atg5 KO MEFs were treated with control, H89 or clofibrate (Clofi) in the absence or presence of nocodazole (Noco). Lipidation reactions with the total membranes from the treated cells were performed. (D–F) The in vitro lipidation of LC3 recovers with restoration of ERGIC. Atg5 KO MEF cells were treated with BFA followed by 100 µM H89. Cells were then washed with fresh medium to remove the drugs and, at indicated intervals, samples were collected for immunofluorescence (D) or total membrane collection for the in vitro lipidation reaction (E). Quantification of the recovery of lipidation activity, ERGIC and Golgi are shown in (F). Bar, 10 µm. Quantification of lipidation activity was shown as the ratio of LC3-II to LC3-I (II/I).