EBP interacts with EEN and Sos2 simultaneously. (A) Yeast transformation assay of EBP with Sos2. Schematic diagram of deletion constructs of EBP is presented. Deletion mutants of EBP cloned in Gal4-BD plasmid were each cotransformed with Sos2 Gal4 DNA-AD 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 Sos2-C was incubated with GST or GST-EBP SH3 domains (SH3 A-B, B-C, or C-D) immobilized on glutathione beads. Binding protein was detected with anti-Myc antibody. (C) Quantitative assay for EBP-EEN interaction. Transformants of Gal4-BD and Gal4-AD fusion pairs were assayed using ONPG substrate. Relative β-galactosidase activity was expressed as arbitrary units. Error bars represent ± SD. (D) Simultaneous interaction of EBP with Sos2 and EEN in HeLa cells. Expressions of EBP and Sos2-C in lysates of HeLa cells transfected with different combinations of Myc-tagged EBP and Sos2-C were confirmed in the upper panel. GST pull-down assay was shown in the lower panel using the HeLa cell lysates shown in the corresponding upper panel. HeLa cell lysates were incubated with GST or GST-EENSH3 fusion protein immobilized on glutathione beads. Bound proteins were detected by anti-Myc antibody.