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Fig. 4. Interaction between the UIMs of Eps15 and Ubiquilin. (A) Cos-1 cells were transiently transfected with GFP-ubiquilin and FLAG-Eps15 wild-type (WT), UIM1mut (E863A/S864A/E865A) or UIM2mut (L883A/L885A). Immunoprecipitation (IP) was performed with anti-FLAG antibody ( ${alpha}$ -FLAG). The immunoprecipitates were subjected to immunoblotting (IB) with anti-GFP antibody ( ${alpha}$ -GFP). The membrane was stripped and reprobed with anti-FLAG antibody. Notice that equal amounts of Eps15 were present in each immunoprecipitate. (bottom) An anti-GFP immunoblot of total cell lysates as a control for GFP-ubiquilin expression. (B) Equal amounts of GST alone, GST-UIM1 (amino acids 847-877 of mEps15) or GST-UIM2 (amino acids 872-897 of mEps15) immobilized on glutathione-Sepharose beads was incubated with total cell lysates (TL, 5% of the total input is shown) of mock-transfected or GFP-ubiquilin transfected Cos-1 cells (Cos-1 + GFP-ubiquilin). The beads were washed and subjected to SDS-PAGE followed by immunoblotting (IB) with anti-ubiquitin antibody ( ${alpha}$ -ubiquitin). In the experiment using GFP/ubiquilin-transfected cells, the membrane was stripped and reprobed with anti-GFP antibody. Notice that the non-ubiquitinated GFP-ubiquilin (100 kDa) was precipitated by GST-UIM1 and, to a lesser extent, by GST-UIM2. (bottom) Coomassie staining of the membranes.

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