Fig. 5. The UBL domain of ubiquilin is sufficient to promote the interaction with Eps15 UIMs. (A) GFP-tagged ubiquilin constructs. UBA, ubiquitin-associated domain (amino acids 493-589); UBL, ubiquitin-like domain (amino acids 14-129). (B) Cos-1 cells were transfected with FLAG-Eps15 and GFP alone, GFP-ubiquilin, GFP-UBL or GFP-UBA. Cell lysates were subjected to immunoprecipitation (IP) with anti-GFP antibody (-GFP) followed by immunoblotting (IB) with anti-FLAG antibody (-FLAG). The membrane was stripped and reprobed with anti-GFP antibody to check the expression level of each GFP construct. Degradation products of the GFP-UBL, GFP-UBA and GFP-ubiquilin are marked with asterisks (^*). (bottom) Anti-FLAG immunoblot of total cell lysates as a control for FLAG-Eps15 expression. (C) Equal amount of GST alone, GST-UIM1, GST-UIM2 or GST-UIM1+2 immobilized on glutathione-Sepharose beads was incubated with purified recombinant His₆-Myc-Ubiquilin or His₆-Myc-UBL (2% and 10%, respectively, of the total input are shown). The beads were washed and subjected to SDS-PAGE followed by immunoblotting (IB) with anti-Myc antibody. (bottom) Representative Coomassie staining of the membrane for both experiments. Notice that both His₆-Myc-Ubiquilin and His₆-Myc-UBL were precipitated more efficiently by GST-UIM1 than by GST-UIM2.