Fig. 2

MDM2 binding is required for ATF3-mediated increase of p53 level. a, b FLAG-tagged V81E, I98E, or V81EI98E was coexpressed with MDM2 in H1299 cells, and subjected to FLAG-IP to determine MDM2-binding activity. H1299 cells transfected with FLAG-ATF3wt or FLAG-ATF3VPI (V81EP83DI98E) in the presence/absence of MDM2 were subjected to co-IP with the MDM2 antibody (c) or the anti-FLAG affinity gel (d) for western blotting as indicated. e A close look at the ATF3 BR structure and the ubiquitination sites. The three residues required for ubiquitination (K106, K107, and K108) are colored in blue. R88G is colored in red. f In vitro-translated FLAG-ATF3VPI and the wild-type control were subjected to in vitro ubiquitination assays. g H1299 cells were transfected as indicated for western blotting