Fig. 1. Identification of an interaction between Eps15 and ubiquilin. (A) The pACT2 construct encoding ubiquilin (amino acids 14-589) fused to the GAL4 transactivation domain was co-transformed with pGBT8 constructs expressing the GAL4 DNA binding domain alone (empty vector) or fused with the EH domains of Eps15 or Eps15R (Eps15-EH or Eps15R-EH) in S. cerevisiae strain AH109. Yeast was grown on selective medium (SC -Trp -Leu -Ade) for 4 days. (B) Cos-1 cells were transiently transfected with constructs encoding GFP-ubiquilin and FLAG-Eps15, Myc-Eps15R or empty vector (-), as indicated. Immunoprecipitation (IP) was performed with anti-FLAG (-FLAG) or anti-Myc (-Myc) antibody. The immunoprecipitates were separated by SDS-PAGE followed by immunoblotting (IB) with anti-GFP antibody (-GFP). The membranes were stripped and reprobed with either anti-FLAG or anti-Myc antibody. Total lysates immunoblotted with anti-GFP antibody are shown as a control for GFP-ubiquilin expression. Notice the ladder-like pattern of ubiquilin in the immunoprecipitates but not in the total lysates.