Fig. 3

MDM2 catalyzes ATF3 ubiquitination at K106, K107, and K108. a In vitro translated MDM2 was incubated with immobilized GST-p53 in the presence/absence of increasing amounts of recombinant ATF3 for GST-pulldown assays. b H1299 cells were cotransfected with myc-MDMX, FLAG-MDM2, and/or ATF3-wildtype (ATF3-wt) for FLAG-IP assays. Bound proteins were subjected to western blotting to detect the MDM2–MDMX binding. c Indicated FLAG-tagged ATF3 C-terminal truncates were in vitro translated, and incubated with or without MDM2 for in vitro ubiquitination assays. Ubiquitinated proteins were detected with the FLAG antibody. d The sequence of ATF3 aa 100–115. The K to R substitutions used in this study are depicted. e FLAG-tagged 5KR was in vitro translated, and incubated with immobilized GST-MDM2 (384–491) for GST-pulldown assays. f Indicated FLAG-tagged ATF3 mutants were in vitro translated for in vitro ubiquitination assays. g FLAG-ATF3wt or 5KR was coexpressed with HA-ub and MDM2 in H1299 cells. Cell lysates were subjected to FLAG-IP followed by western blotting to detect ubiquitinated proteins with the HA antibody. h H1299 cells were transfected as indicated, and subjected to FLAG-IP to determine MDM2-binding activity. i FLAG-ATF3wt or mutants was coexpressed with HA-ub and MDM2 for in vivo ubiquitination assays