Proline-rich motif of EBP is responsible for binding with EEN SH3 domain. (A) Yeast transformation assay of EBP with EEN. Schematic diagram of deletion constructs of EBP is shown. The PRR and SH3 domains are represented by hatched and black boxes, respectively. The arrowheads locate the putative prolinerich binding motifs, PPERP within amino acids 343-347 and PPRPKP within amino acids 400-405. Deletion mutants of EBP cloned in Gal4-AD plasmid were each cotransformed with EEN Gal4-BD plasmid into yeast strain SFY526. Positive interaction accessed by colony lift filter assay is indicated by +. (B) GST pull-down assay. Extract of HeLa cells expressing Myc-tagged EEN was incubated with GST, GST-EBP272-358, or GST-EBP348-419 immobilized on glutathione beads. Binding protein was detected with anti-Myc antibody. (C) Interaction of EBP with EEN in HeLa cells. Lysates of HeLa cells transfected with Myc-tagged EBP1-767 or EBP76-767 and/or FLAG-tagged EEN or SH3 domain deletion mutant, EENΔSH3 FLAG, were subjected to immunoprecipitation (IP) with anti-FLAG antibody followed by anti-Myc immunoblotting (WB). TCL was immunoblotted with anti-Myc antibody. The positions of immunoblotted proteins are indicated by arrowheads. The nonspecific immunoglobulin heavy chain is marked by an arrow.