Fig. 7. VgRBP71 interacts with Prrp. (A) A yeast two-hybrid screen of a Xenopus oocyte cDNA library using the VLE-binding protein Prrp as bait retrieved approximately 15 preys containing VgRBP71. This interaction was characterized using a colony-lift filter assay. Each column represents three independently selected colonies with the following bait-prey combinations. 1, positive control (p53/T antigen); 2, negative control (lamC/T antigen); 3, Prrp/VgRBP71; 4, Prrp/prey vector; 5, bait vector/VgRBP71; 6, RNA binding domain of Prrp (amino acids 1-251)/KH domains of VgRBP71 (amino acids 1-449); 7, RNA-binding domain of Prrp/C terminus of VgRBP71 (amino acids 450-672); 8, proline-rich domain of Prrp (amino acids 242-360)/KH domain of VgRBP71; 9, proline-rich domain of Prrp/C terminus of VgRBP71. (B) The interaction between Prrp and VgRBP71 was also tested using a co-immunoprecipitation assay. VgRBP71, carrying an HA epitope, and Prrp were expressed individually in rabbit reticulocyte lysate; Prrp was labeled with [³⁵S]. Samples of the proteins were incubated together for 1.5 hours and then VgRBP71 retrieved with protein A-Sepharose beads coupled with HA antibody. The immunoprecipitate was analyzed by SDS-PAGE followed by autoradiography. Lane 1, VgRBP71 and Prrp; lane 2, VgRBP71 and Prrp incubated with beads not coupled to HA antibody; lane 3, Prrp alone incubated with beads coupled to HA antibody. (C) Co-immunoprecipitation of VgRBP71 and Prrp from oocyte extract. [³⁵S]-labeled Prrp was injected into stage III/IV oocytes, which were then kept overnight. Oocytes were disrupted manually and incubated with protein A-Sepharose alone (lane 2) or protein A-Sepharose coupled to anti-VgRBP71 antibody (lane 3). The immunoprecipitate was analyzed as described above. Lane 1 contains total extract prepared from injected oocytes and serves as a standard. Arrows indicate the position of Prrp.