EEN SH3 domain mediates interaction with EBP. (A) Schematic representation of deletion mutants of EEN used in the yeast 2-hybrid transformation assay. The hatched box indicates the central coiled coil domain (CC), and the black box denotes the SH3 domain. FL and N represent the full-length and N-terminus of EEN, respectively. (B) Colony lift filter assay. Positive X-gal staining was observed in cotransformants expressing FL/clone no. 30 and SH3/clone no. 30 but not in negative controls, FL/pGADGH, and pAS2-1/clone no. 30. (C) GST pull-down assay. HeLa cells total cell lysate expressing Myc-tagged EBP was incubated with either GST or GST-SH3 (SH3 domain of EEN) immobilized on glutathione beads, and the bound proteins were detected with anti-Myc antibody. TCL indicates 10% of total cell lysate used in the assay.