Establishing a linearly responding [³H]GABA uptake assay in an appropriate cell type. 50, 100, 250, or 500 ng of wild-type mGAT1 plasmid or an equimolar amount of mGAT1₀GFP or pcDNA3.1(+) plasmid was transfected/well. 20-min uptake assays were performed on transfected N2a cells in 2.5 µM (A) or 80 µM (B) [GABA]_O. (C) Identical transfections were performed in HEK 293T cells, and 20-min uptake assays were performed in 2.5 µM of extracellular [GABA]_O. Each point represents the mean of six transfections ± SEM. (D) Comparison of the fluorescence volume density of mGAT1₀GFP expressed in N2a and HEK 293T cells. **, significant difference as determined by Student's t test; P ≤ 0.01. Summed Z-stack images of representative N2a (E) and HEK 293T cells (F) expressing mGAT1₀GFP. Bar, 10 µm. Time course experiments were performed in 2.5 µM (G) or 80 µM (H) [GABA]_O on N2a cells transfected with 100 ng/well of wild-type mGAT1 plasmid or an equimolar amount of the pcDNA3.1(+) plasmid. Each data point represents the mean of six transfections ± SEM.