Fig. 7
The Box II region of p53’s DNA-binding domain (DBD) promotes interaction with Mdm2. a Analyses of domains in p53 required for nutlin-resistant interaction with Mdm2 is summarized here. Locations of the transactivation domain (TAD), the proline rich SH3 domain, the DBD, the tetramerization domain (TD), the regulatory domain (RD), and the highly conserved Boxes (I to V) are indicated in the primary structure of human p53. The p53 constructs indicated by red and green lines are nutlin sensitive and nutlin resistant, respectively, in the Y2H assay with Mdm2. b Mdm2-HA, full-length p53 (p53-FL), p53 (1–160), p53 (1–52), p53 (1–116), and p53 (1–143) proteins were synthesized by in vitro translation. Binding assay was performed by incubating Mdm2-HA with the different p53 variants, followed by immunoprecipitation of Mdm2-HA using an anti-HA antibody and measuring the amount of co-immunoprecipitated p53 by western blotting analysis using anti-p53 (DO-1) antibody. Input (IN), immunoprecipitated proteins (IP), and eluate from control IP performed without Mdm2-HA (CON) were loaded on SDS-PAGE gels. c Amount of Mdm2-HA immunoprecipitated in b was analyzed by western blotting using an anti-HA antibody. IN is diluted five-fold in comparison to the IP samples for the different binding reactions (indicated in red)