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Fig. 3. Participation of clathrin, epsin, and dynamin in trans-endocytosis of CD47. (A-C) HEK293T cells were transfected with the vector siRNAchc-1, which encoded both mouse SHPS-1 and a siRNA specific for human CHC mRNA. Twenty-four hours after transfection, the cells were isolated, plated on new culture dishes, and incubated for 48 hour before the addition of CHO-CD47 cells. After coculture for 1 hour, the cells were fixed and subjected to two-color immunostaining with mAbs to SHPS-1 (green; A) and to CHC (red; B). The merged image is shown in C. Asterisks indicate cells in which CHC was depleted and exogenous SHPS-1 was expressed. Bar, 20 µm. (D) HEK293T cells transfected with siRNAchc-1 or siRNAchc-2 were treated as in A-C and then fixed and stained as in Fig. 1J-L. The cells were also stained with pAbs to SHPS-1 and AMCA-conjugated goat pAbs to rabbit IgG in order to confirm the expression of SHPS-1. As a control, HEK293T cells were transfected with the corresponding SHPS-1 expression vector without the insert for CHC siRNA. The numbers of surface, internalized, and total CD47-positive vesicles in transfected HEK293T cells located adjacent to CHO-CD47 cells were determined by assay B as described in Materials and Methods, with the exception that data were collected from 20 cells in each experiment. (E) CHO-Ras cells were transfected with an expression vector for SHPS-1 and that for the indicated DN mutants of epsin or dynamin 1 (or with the corresponding empty vector). Twenty-four hours after transfection, the cells were cocultured with CHO-CD47 cells for 1 hour. All cells were then fixed and stained as in Fig. 1J-L. They were also stained with mAbs to the Myc or HA epitope tags in order to confirm expression of the epsin or dynamin 1 mutants. The numbers of surface, internalized, and total CD47-positive vesicles in SHPS-1-expressing CHO-Ras cells positioned adjacent to CHO-CD47 cells were determined by assay B. Data in D and E are means ± s.e.m. from three separate experiments. *P<0.05, **P<0.01 versus corresponding control value.
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