Fig. 5.

The transport function (upper panel) and molecular size (lower panel) of N58Q-, N68Q- and N58Q/N68Q-HsPCFT mutants. Upper panel; [³H]MTX influx in HeLa cells transiently transfected with wild-type HsPCFT or glycosylation mutants. Uptake of 0.5 µM [³H]MTX was assessed at pH 5.5 and 37 °C over 2 min. Transport activity in wild-type transfectants is 155.3 ± 36.4 pmol/mg/2 min. The p value reflects the difference in activities of the mutant as compared to the wild-type HsPCFT transfectants. Data are the mean ± SEM from three independent experiments. Lower panel; Western blot analysis of wild-type and glycosylation mutant HsPCFT membrane proteins. An equal amount of lysate (20 µg) was loaded and the blots were probed with a peptide antibody directed to the C-terminus of HsPCFT. The numbers shown on the left side indicate the molecular sizes in the protein ladders. The blot is representative of three separate experiments.