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. 2004 Feb 2;164(3):461–470. doi: 10.1083/jcb.200311093

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Copyright © 2004, The Rockefeller University Press

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Figure 3. — Growth factor stimulates exocytosis-dependent up-regulation of Rap1 on the PM. (A) COS-1 cells were transfected with GFP-Rap1 wild type without (top and middle) or with cotransfection of dominant-negative Rab11BP (bottom). Cells were serum starved 24 h after transfection, stimulated with EGF in the absence (top and bottom) or presence (middle) of NEM, and imaged before and 5 min after stimulation. Arrow indicates areas of GFP-Rap1 up-regulation on PM ruffles. PM up-regulation of Rap1 was observed in all control cells and validated by measurement of relative fluorescence intensity as described in Materials and methods. In contrast, only 16 ± 9% and 8 ± 8% (mean ± SEM) of NEM-treated and dominant-negative Rab11BP (DN Rab11BP) transfected cells, respectively, showed up-regulation on PM (n = 4; P < 0.0001 for each condition compared with control). (B) COS-1 cells expressing GFP-H-Ras that were serum starved and stimulated as in A showed no change in Golgi apparatus (arrowhead) or PM (arrow) expression. Images shown are representative of seven Z slices acquired to compensate for minimal focal drift. Bars, 10 μM.