Fig. 3.

Phosphorylation of ULK1 plays a key role in fusion of autophagosomes to lysosomes. a The control vector, ULK1 WT, S423A, and S423D were transfected into the ULK1/2-KO/D cells and treated with/out Chloroquine (CQ, an lysosomal inhibitor, 20 nM). The lysate was analyzed by WB with LC3, p62 and actin antibodies. b The quantified ratio (CQ-ULK1s/ULK1s) of LC3-Π and p62 is shown. c Representative confocal images of ULK1/2-KO/D HeLa cells transfected with Myc-ULK1 WT and mutants treated with/out CQ shown as the average of puncta per cell. Scale bar, 10 μm. A minimum of 20 cells were counted. d The quantified ratio (CQ-ULK1s/ULK1s) of puncta per cell is shown. e Representative confocal images of in vitro fusion assay. Lysosomes (green) and autophagosomes (red) are mixed with purified GST-ULK1 WT, S423A, and S423D. Scale bar, 5 μm. The fusion percentage was quantified by comparison of the number of yellow dots to the number of red dots and yellow dots. All values are means ± SEM of three independent experiments. Student’s t test (unpaired); NS: no significance; *p < 0.05, **p < 0.01, and ***p < 0.001