Fig. 3. VGLUT3 functions as a vesicular glutamate transporter. (A) hVGLUT3 protein is expressed in hVGLUT3-transfected cells, but not in mock-transfected cells. Triton X-100 extracts from each cell line (50 µg/lane) were analysed by western blotting using hVGLUT3-specific antibody. A broad band with a molecular mass of ∼70 kDa was detected in hVGLUT3 cell extracts only. (B) Time course of glutamate accumulation in hVGLUT3-transfected (filled circles) and mock-transfected (open circles) BON cells. The membranes prepared from hVGLUT3-transfected cells accumulated ∼2-fold higher amounts of glutamate than those from mock-transfected cells. (C) Glutamate uptake by hVGLUT3 is driven by proton-electrochemical gradient as transport activity was reduced by the proton uncoupler, FCCP (46 µM), and the vacuolar H-ATPase inhibitor, bafilomycin A₁ (100 nM). Error bars are the standard error of the mean (n = 3).