Figure 7.

Epsin and Eps15/Eps15R are involved in DAT endocytosis and coimmunoprecipitated with DAT. A, HeLa/YFP-HA-DAT cells were mock transfected or transfected with the mixture of epsin-1 and Eps15 siRNA SMARTpools, or epsin-1 siRNA duplex 2, Eps15 siRNA duplex 3 (Huang et al., 2004), and Eps15R SMARTpool, or with a mixture of SMARTpools of all three siRNAs using conventional forward transfection. Cell lysates were probed with antibodies to Eps15, Esp15R, and epsin-1 to determine the extent of depletion of proteins, and with antibodies to an unrelated protein (PP1) to control for sample loading. B, HA11 endocytosis was examined as in Figure 2. Identical intensity settings for Cy5 or Cy3 fluorescence were used to generate images of mock- versus specific siRNA-transfected cells. The sum projection images of three consecutive optical sections are presented. Scale bars, 10 μm. Asterisks indicate positions of cell nuclei. C, YFP-HA-DAT was immunoprecipitated from parental HeLa or HeLa/YFP-HA-DAT cells (±PMA), and the immunoprecipitates and lysates were probed with antibodies to Nedd4–2, Eps15 and epsin-1, and then with antibodies to DAT.