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Fig. 5. Regulation of trans-endocytosis of CD47 by Rac, Cdc42 and Rab5. (A) CHO-Ras cells were cotransfected with an expression vector for SHPS-1 and with a vector for either Rac(T17N) or NWASP-CRIB (or with the corresponding empty vector). Twenty-four hours after transfection, the cells were cocultured with CHO-CD47 cells for 1 hour. CHO–SHPS-1 cells were also treated with 0.5 µM cytochalasin D (CytoD) or with dimethyl sulfoxide (DMSO) vehicle for 30 minutes before coculture with CHO-CD47 cells. All cells were then fixed and stained as in Fig. 1J-L. The numbers of surface, internalized, and total CD47-positive vesicles in SHPS-1-expressing cells adjacent to CD47-expressing cells were determined by assay B. Cells were also stained with a mAb to the Myc epitope in order to confirm the expression of Myc-tagged Rac(T17N) or NWASP-CRIB. Data are means ± s.e.m. from three separate experiments. *P<0.05, **P<0.01 versus the corresponding control value. (B-D) CHO–SHPS-1 cells were transfected with an expression vector for Rab5(Q79L; C) or for Rab5(S34N; D). Twenty-four hours after transfection, the cells were cocultured with CHO-CD47 cells for 1 hour, fixed, and subjected to immunostaining with mAbs to CD47 (red; B-D) and to the HA epitope tag of the Rab5 mutants (green; C,D). In a control experiment (B), CHO–SHPS-1 cells were transfected with a vector for GFP and then analyzed as for the cells expressing the Rab mutants, with the exception that GFP was detected on the basis of its intrinsic fluorescence (green). Bar, 20 µm.
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