Fig. 5: TMEM189 disrupts the interaction between ULK1 and TRAF6, impaires K63-linked ULK1 polyubiquitination.

a HEK293T cells were transfected with indicated plasmids for 24 h, then treated with or without 50 μM of CQ for 4 h. Cell lysates were subjected to IP using an anti-GFP. The endogenous ULK1 and GFP-TRAF6 proteins were detected in the immunoprecipitates by western blotting. b HEK293T cells were cotransfected with indicated plasmids for 24 h, cell lysates were subjected to IP using an anti-GFP. The FLAG-TMEM189 and GFP-TRAF6 proteins were detected in the immunoprecipitates by western blotting. c ULK1^+/+ and ULK1^−/− HeLa cells were transfected with FLAG-vector or FLAG-TMEM189 for 24 h, then cell lysates were subjected to IP using an anti-FLAG. Endogenous TRAF6 and FLAG-TMEM189 proteins were detected in the immunoprecipitates by western blotting. d TRAF6^+/+ and TRAF6^−/− HEK293T cells were transfected with indicated plasmids for 24 h, then cell lysates were subjected to IP using an anti-GFP. FLAG-TMEM189 and GFP-ULK1 proteins were detected in the immunoprecipitates by western blotting. e HEK293T cells were transfected with indicated plasmids for 24 h, then cell lysates were subjected to IP using an anti-GFP. FLAG-TMEM189, FLAG-TRAF6 and GFP-ULK1 proteins were detected in the immunoprecipitates by western blotting. f The stable shcontrol- or shRNF115-expressing HEK293T cells were cotransfected with indicated plasmids for 24 h, then cell lysates were subjected to IP using an anti-GFP. FLAG-TRAF6 and GFP-ULK1 proteins were detected in the immunoprecipitates by western blotting. g The cotransfection of HEK293T cells were shown in the figure. After 24 h, these cells were treated with MG132 for 6 h, then the cell lysates were immunoprecipitated with an anti-GFP, and the western blotting was probed with an anti-FLAG antibody to detect ubiquitinated GFP-ULK1 (left panel). Simultaneously, 10% cell lysates were used to western blotting (right panel). h The stable shcontrol- or shRNF115-expressing HEK293T cells were cotransfected with indicated plasmids for 24 h, then treated with MG132 for 6 h. The cell lysates were immunoprecipitated with an anti-GFP, and the western blotting was probed with an anti-FLAG antibody to detect ubiquitinated GFP-ULK1. i HEK293T cells were transfected with indicated plasmids for 24 h, then cell lysates were subjected to IP using an anti-GFP. HA-ULK1, GFP-ULK1 and FLAG-TMEM189 proteins were detected in the immunoprecipitates by western blotting. Simultaneously, 10% cell lysates were used to western blotting.