Fig. 4
From: ULK1 phosphorylates Sec23A and mediates autophagy-induced inhibition of ER-to-Golgi traffic
ULK1 phosphorylates Sec23 and changes its binding to other COPII components. a Myc-ULK1 WT was co-transfected with indicated COPII components (GFP-tag) pairwise in HEK293T cell. ULK1 was immunoprecipitated by anti-Myc antibody and the presence of co-precipitated COPII components were detected by blotting with anti-GFP antibody. b Myc-ULK1 and Myc-Sec23A were separately overexpressed and purified from HEK293T cells by immune-isolation using anti-Myc antibody. Some of ULK1 transfected cells were put under amino acid starvation to further activate the ULK1 kinase activity. Beads loaded with ULK1 and Sec23A protein were mixed to allow ULK1 to phosphorylate Sec23A in vitro. Phosphorylated Sec23a was detected by Mouse anti-serine/threonine antibody. Total Sec23a was detected by Rabbit anti-Sec23a antibody. c-e HEK293T cells were transfected with the indicated plasmids and treated by EBSS or complete medium prior to harvest. AA star. Means amino acid starvation. After harvest, Sec23A was immunoprecipitated and detected by using monoclonal anti-GFP antibody. Sec31A (c), Sar1b (d) and Sec24D (e) were detected by mouse anti-Myc antibody