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. 2019 Aug 5;10(8):583. doi: 10.1038/s41419-019-1805-9

Fig. 6. TOPK phosphorylates ULK1 at Ser469, Ser495, and Ser533, inhibiting its activity and reducing its stability.

Fig. 6

a PRK5-HA-ULK1-WT and PRK5-HA-ULK1-Ser469/495/533AAA were transiently transfected into HEK293T cells as indicated. ATG13, p-ATG13, Beclin-1, p-Beclin-1, HA, and Tubulin were analyzed using western blots. b PRK5-HA-ULK1-WT and PRK5-HA-ULK1-Ser469/495/533AAA were transiently transfected into HEK293T cells, 48 h later, cells were extracted, immunoprecipitated with anti-HA, and probed with anti-FIP200, anti-ATG101, and anti-ATG13 by western blots. Equal protein loading and transfection efficiency were determined by western blots using the total extracts. c PRK5-HA-ULK1-WT and PRK5-HA-ULK1-Ser469/495/533AAA were transiently transfected into HEK293T cells as indicated. Then cells were treated with EGF (20 ng/ml) for 15 min, followed by the addition of CHX (100 μg/ml) for 6, 8, and 12 h before extraction. HA and Tubulin were analyzed using western blots (left). Data of c represent the results of triplicate experiments. The densities of HA/Tubulin (right) were determined by Image J. The data were presented in the form of mean ± SD, *P < 0.05, **P < 0.01, ***P < 0.001. d HA-ULK1-WT or HA-ULK1-AAA, Flag-ubiquitin, and GAL4-TOPK were cotransfected into HEK293T cells, 48 h later, cells were extracted, immunoprecipitated with anti-HA, and probed with anti-Flag, anti-FIP200, anti-ATG101, and anti-ATG13 by western blots. Equal protein loading and transfection efficiency were determined by western blots using the total extracts. e PRK5-HA-ULK1-WT and PRK5-HA-ULK1-Ser469/495/533AAA were transiently transfected into HEK293T cells as indicated. Cells were harvested 48 h later. LC3B, HA, and Tubulin were analyzed using western blots. f PRK5-HA-ULK1-WT and PRK5-HA-ULK1-Ser469/495/533AAA were transiently transfected into HEK293T cells as indicated. 24 h later, cells were starved for 24 h, then rapamycin (50 nM) and CQ (50 μM) were added, respectively, for 15 min and 1 h. LC3B, HA, and Tubulin were analyzed using western blots. The densities of LC3-II/Tubulin (Fig. 6f, bottom) were determined by Image J. The data were presented in the form of mean ± SD, *P < 0.05, **P < 0.01, ***P < 0.001. Data represent the results of triplicate experiments