Fig. 1

The ATF3 basic region is required for p53 stabilization. a Schematic representation of the ATF3 domains responsible for binding to MDM2 and p53. Red two-direction arrows indicate interactions. Positions of lysine residues are also marked as black ovals. b H1299 cells were transfected with p53, GFP, in the presence/absence of FLAG-tagged ATF3wt, JDP2, or ATF3 deletions as indicated for western blotting. The GFP level was determined for the control of transfection efficiency. c The sequence of ATF3 BR is compared with that of JDP2. d ATF3 and JDP2 structures predicted with the I-TASSER server. Note the similarity and the difference in the BR region (colored with red) between ATF3 and JDP2. The side chains of V81 and I98 are shown in green. P83 is colored in yellow. e FLAG-tagged ATF3(1–101) or JDP2 was in vitro translated, and incubated with immobilized GST-MDM2 (384–491) for GST-pulldown assays. The MDM2 C-terminal fusion was used in most of the experiments because it was better folded and expressed in E. coli. ATF3 efficiently binds to this C-terminal fragment [21]. f In vitro translated p53 was incubated with immobilized JDP2 or ATF3 for GST-pulldown assays. Arrows indicate GST or GST fusion proteins. g In vitro translated FLAG-ATF3 or FLAG-JDP2 was incubated with purified MDM2 and other ubiquitination reaction components for in vitro ubiquitination assays. Ubiquitinated proteins were detected with the FLAG antibody. h Indicated FLAG-tagged proteins were in vitro translated and incubated with GST-MDM2 (384–491) for GST-pulldown assays. i H1299 cells were transfected as indicated, and subjected to FLAG-IP for western blotting to detect MDM2 binding. j Indicated proteins were in vitro translated and subjected to in vitro ubiuquitination assays. k H1299 cells were transfected as indicated for western blotting