Fig. 6.

Effect of 1 mM MTSEA on [³H]Gly-Sar uptake activities of the cysteine-scanning mutants of TMD10 of hPEPT1. At 72 h post-transfection, the transfected cells adhered to the wells were washed with the transport medium (MES-Tris, pH 6 buffer). Each well was then incubated for 10 min at 37°C with a solution containing [³H]Gly-Sar (0.5 μCi/ml) after pre-incubation with 1 mM MTESA for 10 min. After washing three times in ice-cold HEPES-Tris, pH 7.4 buffer, the cells were lysed in 500 μl of lysis buffer. BCA protein assay reagents were used to determine the protein content of each well, and the cell-associated radioactivity was measured in a Beckman liquid scintillation counter. Results represent the % Gly-Sar uptake of each mutated transporter compared with wild type hPEPT1 (n=4–7). The background uptake values of mock-transfected HEK293 cells were subtracted. The grey bars represent uptake activity in the absence of 1 mM MTSEA, and the black bars represent uptake activities in the presence of 1 mM MTSEA. *, highly significant inhibition of uptake activity by 1 mM MTSEA.