Figure 4.

 In vitro binding of NPF-containing proteins and peptides to Eps15. (A) In vitro binding of Eps15 to Gst fusions of EH-binding proteins. Total cellular lysates from NIH–3T3 cells (1 mg/lane) were incubated with the indicated GST fusion proteins (10 μg) for 1 hr at 4°C. Specifically bound Eps15 was detected by immunoblot with an anti-Eps15 antibody. (B) Binding of Eps15 to GST fusions encompassing NPF-containing peptides. GST fusion proteins were engineered to encompass the NPF-containing peptides underlined in Fig. 1C and are indicated with the names of the proteins from which the peptides were derived. In vitro binding experiments were performed as described in A. Lanes marked LYS in A and B were loaded with 50 μg of total cellular proteins to serve as a reference for the position of Eps15. The position of Eps15 is also indicated.