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Fig. 6. Effects of overexpression of Rab18 in COS7 cells. (A) Colocalization of GM130 and COPII in immunofluorescence images of cells expressing moderate levels of GFP-Rab18. GM130 was visualized using a Cy3-tagged secondary antibody (red), COPII using Cy5 (blue). (B) Colocalization of GM130 and COPII staining in cells overexpressing very high levels of GFP-Rab18 staining was as in A. Magnifications of boxed areas are shown below merged images in A and B. (C,D) Trafficking of VSVG in cells strongly expressing Rab18 then incubated at 40°C for 48 hours to accumulate VSVG-YFP in the ER. Cells were then fixed and stained with VG antibody without permeabilization to detect cell-surface VSVG. (C) Singly transfected cells (VSVG-YFP only) were incubated at 32°C for 3 hours to allow exit from the ER. (D) Cells double-transfected with VSVG-YFP and the indicated Rab18 construct after shifting to 32°C for 3 hours. (E) Uptake of Cy3-EGF (100 ng/ml; for 30 minutes following a 2 hour serum starvation) in COS7 cells expressing GFP-Rab18 for 48 hours. Scale bars: 10 µm. (F) Quantification of the VSVG trafficking experiment in D. Each measurement is an average of >100 cells. (G) Quantification of endocytosis of Cy3-Tf (25 µg/ml; for 30 minutes following 2 hours of serum starvation) or EGF in cells expressing the indicated constructs for 48 hours. >100 cells were averaged for each sample. Data are mean ± s.e.m.
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