Fig. 2. Ubiquitinated GFP-ubiquilin interacts with Eps15 but ubiquitination is not essential for this interaction. (A) Cos-1 cells were transiently transfected with a vector encoding GFP-ubiquilin or GFP alone, or mock transfected. Cell extracts were subjected to immunoprecipitation (IP) with anti-GFP antibody (-GFP) in RIPA buffer. Immunoprecipitates were separated by SDS-PAGE and immunoblotted (IB) with anti-ubiquitin antibody (-ubiquitin). The membrane was stripped and reprobed with anti-GFP antibody. (B) Cos-1 cells were transiently co-transfected with FLAG-Eps15 and GFP-ubiquilin or empty vector (-), as indicated. Immunoprecipitation (IP) was performed with anti-FLAG antibody (-FLAG). The immunoprecipitates were separated by SDS-PAGE followed by immunoblotting (IB) with anti-ubiquitin antibody. The membrane was stripped and reprobed with anti-GFP antibody. After a second stripping, the membrane was reprobed with anti-FLAG antibody. (C) Equal amounts of Cos-1 total cell lysate expressing FLAG-Eps15 (TL; 5% of the total input is shown) was incubated with GST or GST-ubiquilin immobilized on glutathione-Sepharose beads. The beads were washed and subjected to SDS-PAGE followed by immunoblotting (IB) with anti-FLAG antibody. The lower panel shows a Coomassie staining of the membrane. The positions of GST and GST-ubiquilin are indicated.