Fig. 3. Localization of newly synthesized GM130 and GRASP65 in the presence of a dominant negative form of Sar1p. (A) Sar1p H79G mutant (mSar1p; top panels) or buffer (mock; bottom panels) were first microinjected into the cytoplasm of NRK cells with cascade-blue-conjugated BSA as an injection marker. After 15 minutes incubation, plasmids encoding NAGFP and FLAG-GM130 were subsequently microinjected into the nuclei and the cells were further treated as described in Materials and Methods. Two-dimensional projections of triple range confocal microscope images are shown: NAGFP (GFP fluorescence; right panels), FLAG-GM130 (Cy3 staining; middle panels) and giantin (Cy5 staining; left panels). Asterisks indicate the microinjected cells. (B) As (A) except that GRAP65-HA was expressed in the middle panels. (C) mSar1p was microinjected into the cytoplasm of NRK cells with cascade-blue-conjugated BSA as an injection marker. After 100 minutes incubation, cells were fixed and subjected to the immunofluorescence staining. Top panels: giantin (left) and GM130 (right) were double stained. Bottom panels: mannosidase II (left) and GM130 (right) were double stained.