Fig. 4.

The effect of tunicamycin treatment on the transport function (upper panel) and the apparent molecular size (lower panel) of HsPCFT. Upper panel; [³H]MTX influx in Hela cells transiently transfected with HsPCFT cDNA and treated with tunicamycin. Uptake of 0.5 µM [³H]MTX was assessed at pH 5.5 and 37 °C over 2 min in HeLa cells grown in the absence or presence of tunicamycin for 48 h. The transport activity in control cells is 214.0 ± 30.2 pmol/mg/2 min. The p value reflects the comparison of the difference between control cells and cells treated with 1 μg/mL tunicamycin. Data are the mean ± SEM from three independent experiments. Lower panel; Western blot analysis of HsPCFT protein after tunicamycin treatment of HeLa cells. Equal amounts of lysate (30 µg) from HeLa cells, grown in the presence of increasing tunicamycin concentrations were loaded and the blots were probed with a peptide antibody directed to the C-terminus of HsPCFT. The numbers on the left indicate the molecular sizes in the protein ladders. The blot is representative of three separate experiments.