Fig. 3: TMEM189 interacts with ULK1 via its N-terminal and knockdown of TMEM189 increases ULK1-ATG13 interaction.

a, b HEK293T cells were cotransfected with GFP-ULK1 and FLAG-TMEM189 plasmids for 24 h, then cell lysates were subjected to IP using an anti-FLAG, an anti-GFP or a control IgG as indicated. GFP-ULK1 and FLAG-TMEM189 proteins were detected in the immunoprecipitates by western blotting. c HEK293T cells were transfected with indicated plasmids for 24 h, then cell lysates were subjected to IP using an anti-FLAG. Endogenous ULK1 proteins were detected in the immunoprecipitates by western blotting. d HeLa cells were incubated with or without EBSS for indicated time, then cell lysates were subjected to IP using an anti-ULK1 or a control IgG. The endogenous ULK1 and TMEM189 proteins were detected in the immunoprecipitates by western blotting. e Construction of truncated GFP-ULK1 plasmids. f HEK293T cells were cotransfected with indicated plasmids for 24 h, then cell lysates were subjected to IP using an anti-GFP. GFP-ULK1 and TMEM189-MYC proteins were detected in the immunoprecipitates by western blotting. g GST-TMEM189^1-45, GST-TMEM189^150-230, GST-TMEM189^183-270 fusion protein and the GST protein were purified and immobilized on Glutathione-Sepharose beads, then incubated with HEK293T cell lysates containing GFP-ULK1. Proteins retained on Glutathione-Sepharose were then blotted using the indicated antibodies. h Purified GST-TMEM189^1-45 and GST protein were incubated with HEK293T cell lysates with or without EBSS incubation for 30 min, the endogenous ULK1 and GST-fusion protein were detected in the washed beads by western blotting. i ULK1 wild-type (ULK1^+/+) and ULK1 knockout (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 ATG13, ATG101 and FLAG-TMEM189 proteins were detected in the immunoprecipitates by western blotting. j The stable shcontrol- or shTMEM189-expressing HeLa cells were transfected with GFP-ULK1 plasmids. Cell lysates were immunoprecipitated using an anti-GFP and analyzed by immunoblotting with anti-ATG13 antibody. Simultaneously, 10% cell lysates were used to immunoblotting.