Figure 5.

Transfected NPY-mRFP is expressed into DCVs only in astrocytes cotransfected with REST/DBD-GFP. The insets of all panels show the positivity of the cells for GFP (also positive for mRFP), documenting that they had been transfected with REST/DBD-GFP or the control construct. (A and B) In control cells, the distribution of NPY-mRFP is primarily in thin cisternae and few puncta (A, deconvolved image), which TIRF shows to be largely excluded from the surface layer of the cytoplasm illuminated by the evanescent wave (B). (C and E) In cells cotransfected with REST/DBD-GFP, NPY-mRFP is concentrated in scattered discrete puncta (C, deconvolved image) and colocalized with Sg2 (E, confocal image), corresponding, therefore, to the DCVs illustrated in Fig. 4 (D and E). The yellow line marks the contours of the cell. (D) Small NPY-positive puncta are abundant in the surface layer of the cytoplasm illuminated by the evanescent wave as illustrated by the TIRF image shown. The large fluorescent structures of E in the peri-Golgi area are accumulations of NPY-mRFP negative for Sg2 (not depicted; also see Fig. S2, E–H). The bottom box highlights the area of the three panels to the right showing the colocalization of NPY-mRFP and endogenous Sg2. A–E are representative of ≥10 cells produced in at least three transfection experiments. Bars: (A–E, left) 15 µm; (E, right) 5 µm.