Fig. 6. Vglut1 increases the steady-state vesicular zinc content. Untransfected PC12 cells (wt), clones carrying either Vglut1 (Vgl), Vglut1 and ZnT3 (VglZn) or the VAMP II N49A mutant were zinc loaded and stained with zinquin. Zinquin fluorescence was determined by flow cytometry (A,C-E). (B) Immunoblot analysis of the expression levels of ZnT3 and Vglut1 in the cell types used in A,C-D. (A-C) Expression of Vglut1 (Vgl) increased zinquin fluorescence by 22%, comparing wild-type (wt) and Vgl cells. Zinquin fluorescence was further increased to 170% and 196% in two different clones co-expressing Vglut1 and ZnT3 (VglZn4 and VglZn6, respectively). No difference in zinquin fluorescence was observed in a PC12 clone carrying only ZnT3 (ZnT3-4). The graph depicts the average mean values of flow-cytometry profiles. Data were normalized with respect to untransfected PC12 cells (100%). `NS' is the background fluorescence determined in unstained cells. (D) Zinc loading was carried out in the absence or presence of bafilomycin A1 and zinc content was determined by zinquin fluorescence and flow cytometry. Bafilomycin A1 decreases zinquin fluorescence in Vgl and VglZn clones. Values are normalized to those obtained in the absence of drug (100%) for each cell type. Average ± s.e.m. Absent error bars are under the graphing threshold. (E) Representative zinquin flow-cytometry profile of untransfected PC12 cells (wt) and a PC12 clone carrying the VAMP II N49A mutant (n=7).