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Fig. 3. The coiled-coil domain and the third domain of Eps15 are necessary for the interaction with ubiquilin. (A) The FLAG-tagged Eps15 deletion mutants. The three structural domains of Eps15 (I, II and III) were deleted as indicated. The FLAG tag (black box) is located at the N-terminus of the constructs. AP-2, AP-2 binding sites; PRM, proline-rich motif; UIMs, ubiquitin-interacting motifs. (B) Cos-1 cells were transiently co-transfected with GFP-ubiquilin and the indicated FLAG-Eps15 constructs (WT, wild type) or empty vector (-). Immunoprecipitation (IP) was performed with anti-FLAG antibody ( ${alpha}$ -FLAG) and samples were subjected to immunoblotting with anti-GFP antibody ( ${alpha}$ -GFP). The membrane was stripped and reprobed with anti-FLAG antibody. Notice that the FLAG-Eps15 constructs are expressed at similar levels. (bottom) Total cell lysates immunoblotted with anti-GFP antibody as a control for GFP-ubiquilin expression.

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