Cdc15 SH3 domain is necessary and sufficient for interaction with C2 domain protein Fic1. (A) Schematic representations of the domains of Cdc15 (top) and Fic1 (bottom). Domains are as indicated, except the PEST domain of Cdc15 is the white oval, and PXXP motifs in Fic1 are indicated with asterisks. Fragments of cdc15 and fic1 from the two-hybrid screen are depicted. (B and C) Coimmunoprecipitation of endogenously tagged Fic1-FLAG₃ and Cdc15-HA₃ from asynchronous cell lysate. Asterisks indicate a background band present in FLAG immunoblots. (D) Coimmunoprecipitations were performed in which Cdc15 or Cdc15_ΔSH3 was isolated using anti-Cdc15 serum, or Fic1-FLAG₃ was isolated with the anti-FLAG antibody, and bound proteins were identified by immunoblotting with either the anti-FLAG antibody or anti-Cdc15 serum. (E) Recombinant MBP or MBP-Cdc15SH3 was purified and incubated with recombinant bead-bound His₆ or Fic1-His₆. Beads were washed, and bound proteins were run on SDS-PAGE and detected by Coomassie staining. One tenth of each MBP protein input is shown as a control. IP, immunoprecipitation; WB, Western blot.