ZBP-89 interacts with p53 in vivo and in vitro. (A) Coimmunoprecipitation of p53 and ZBP-89 from mock-infected AGS cells (lane 1) or from cells infected with Ad5-β-gal (lane 2) or Ad5-ZBP-89 (lane 3). After cell lysis, immunoprecipitation (IP) was performed with the polyclonal p53 antibody followed by immunoblotting with p53 and Flag M2 antibodies. (B) Immunoprecipitation of ZBP-89 and p53 was performed with in vitro-transcribed and -translated ZBP-89 and p53 proteins. Lane 1, input of [35S]methionine-labeled ZBP-89; lane 2, [35S]methionine-labeled p53; lane 3, unlabeled ZBP-89 incubated with radiolabeled p53 and immunoprecipitated with control rabbit serum; lane 4, unlabeled ZBP-89 incubated with radiolabeled p53 and immunoprecipitated with rabbit ZBP-89 antibody; lane 5, unlabeled p53 incubated with radiolabeled ZBP-89 and immunoprecipitated with control rabbit serum; lane 6, unlabeled p53 incubated with radiolabeled ZBP-89 and immunoprecipitated with rabbit anti-p53 antibody. PI, preimmune serum. (C) Mapping of the p53-interacting domain of ZBP-89. The various ZBP-89 constructs used in GST pull-down assays are shown. The ZBP-89 GST fusion proteins were incubated with [35S]methionine-labeled, in vitro-translated p53, pelleted, and then analyzed by SDS-PAGE. Lane 1, input of p53; lane 2, GST alone. (D) Mapping of the ZBP-89-interacting domain of p53. The various p53 constructs used in GST pull-down assays are shown. These proteins were incubated with [35S]methionine-labeled, in vitro-translated ZBP-89 and analyzed as described above for p53. Lane 1, input of labeled ZBP-89; lane 2, GST alone. (E) GST pull-down was performed in the presence of 10 μg of ethidium bromide/ml, and blotting was performed with the M2 anti-Flag antibody. (F) Schematic representation of the ZBP-89 and p53 interaction domains. ZBP-89 interacts with amino acids 160 to 393 of p53. p53 contains an activation domain (AD), DNA binding domain (DNA BD), tetramerization domain (TD), and regulatory domain (RD). p53 binds to the zinc finger DNA binding domain of ZBP-89 (amino acids 154 to 300). ZBP-89 contains an acidic domain (AD), zinc finger DNA binding domain, basic domain (BD), and C-terminal domain (C-TER).