Dynamics of ULK1 and Ub in autophagy against transfected bead. (S3–S7) NIH3T3 cells stably expressing mStr-Ub (magenta) and GFP-tagged (green) LC3 (S3), Atg5 (S4), WIPI-1 (S5), Atg14L1 (S6), or ULK1 (S7) were transfected with Effectene-coated latex beads for 30 min. Live cells were observed at 1-min intervals using a microscope (model IX81; Olympus) equipped with a xenon lamp and cooled charge-coupled device camera (CoolSnap HQ; Roper Scientific) under the control of MetaMorph software. Time is shown in h:min:s. We excluded Atg9L1 in this experiment because Atg9L1 localized to the trans-Golgi network and early endosomes under steady-state conditions, which cannot be distinguished from recruitment to bead-containing endosomes (Young et al., 2006; Kageyama et al., 2011).
