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Fig. 8. Co-expression of zinc and glutamate transporters increases the steady-state vesicular glutamate content in whole cells. Untransfected PC12 cells (wt) or transfected either with Vglut1 (Vgl) or co-transfected with Vglut1 and ZnT3 (VglZn) were stained with glutamate (A,B,D,E) or Vglut1 antibodies (C). (A) Glutamate staining was mainly vesicular and was abolished by the BGG. Glutamate cellular content (B,D) increased in the presence of Vglut1 (Vgl) and co-expression of ZnT3 and Vglut1 further increased the cellular levels of glutamate (VglZn clones 4 and 6). NS, unstained cells; –Ab, cells stained only with the secondary antibody. (C) VglZn4 clonal cells produce less Vglut1 transporter than Vgl cells. (E) PC12 cells or clonal variants were treated in the absence or presence of bafilomycin A1 before antibody staining. Values are normalized to those obtained in the absence of drug (100%) for each cell type. (F) The vesicular index, representing vesicular glutamate pools, was 1.6 times greater in VglZn than in Vgl cells. Average ± s.e.m. Absent error bars are under the graphing threshold.
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