MTX influx kinetics mediated by D156 PCFT mutants. (A) [³H]-MTX influx kinetics mediated by D156G and D156S PCFT, compared with wild-type PCFT. Influx was assessed at pH 5.5 at 37°C over 1 minute with [³H]-MTX concentrations of 0.1, 0.2, 0.5, 1, 2.5, 5, and 10μM. Data are the mean ± SEM from 3 independent experiments. (B) Western blot of biotinylated protein obtained from cells transfected with wild-type PCFT and the D156G and D156S mutants. Wild-type PCFT was diluted to more accurately quantify, by comparison, levels of mutant PCFTs. Ten μL of the biotinylated wild-type PCFT protein fraction was added to 10, 20, 25, and 30 μL of 2× SDS-loading buffer, described as 1:2, 1:3, 1:3.5, and 1:4 dilutions, respectively. The data are representative of 2 independent experiments. A vertical line has been inserted to indicate a repositioned gel lane.