Fig. 1. MDM2 mediates the association between nucleostemin and p53 via the central domain of MDM2 and the coiled-coil and acidic domains of nucleostemin. (A1) Triple-coimmunoprecipitation assays of HA-tagged nucleostemin (NS), Flag-tagged MDM2 and Myc-tagged p53 show that nucleostemin, MDM2 and p53 coexist in the same protein complex. (A2) Binding between nucleostemin and p53 is significantly reduced without MDM2 coexpression. (B) In vivo binding of endogenous nucleostemin and MDM2 is confirmed by coimmunoprecipitation assays, which immunoprecipitated MDM2 or nucleostemin complexes from U2OS cells. (C) MDM2 (top) and nucleostemin (bottom) deletion mutants. Gray lines indicate the deleted regions. (D) Coimmunoprecipitation assays showed that nucleostemin fails to bind MDM2 mutants with deleted I1 or AZ domains (D1), and that deleting the A domain or the BC domain of nucleostemin abolishes its ability to bind MDM2 (D2). Abbreviations: A, acidic domain; AZ, acidic/zinc finger; B, basic domain; C, coiled-coil domain; G, GTP-binding domain; I, intermediate; N, p53-binding domain; R, RING finger; Sup, supernatant; WB, western blot.