Fig. 4

ATF3 mutants devoid of ubiquitination fail to increase the p53 expression level. a Histidine-tagged ATF3wt, K107R, and K108R were purified with Ni⁺-NTA agarose, and their purity was determined by SDS-PAGE. b In vitro translated p53 was incubated with recombinant ATF3wt, K107R, or K108R for in vitro ubiquitination assays. After determining the p53 ubiquitination level using the p53 antibody, the same blot was stripped and incubated with the ATF3 antibody to determine the ATF3 ubiquitination level. c H1299 cells were transfected with FLAG-tagged ATF3wt, ATF3 mutants, HA-ub, and MDM2 as indicated for IP with the HA antibody. The p53 ubiquitination level was determined with the p53 antibody. d Transfected H1299 cells were subjected to FLAG-IP followed by western blotting to determine the p53-binding activity. e H1299 cells were transfected as indicated for western blotting. f H1299 cells were transfected with p53 and MDM2 in the presence/absence of ATF3wt or mutants for western blotting