Fig. 6.

The effect of HA tagging on the transport function (upper panel) and molecular size (lower panel) of the wild-type and deglycosylated (N58Q/N68Q) HsPCFT. Upper panel; [³H]MTX influx in HeLa cells transiently transfected with cDNAs of N- or C-terminus HA-tagged wild-type and N58Q/N68Q-HsPCFT mutant (degly-HsPCFT). Influx 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 288.8 ± 12.6 pmol/mg/2 min. The p value reflects the difference in activity between the wild-type and deglycosylated HsPCFT. Data are the mean ± SEM from three independent experiments. Lower panel; Western blot analysis of membrane proteins from HeLa cells transiently transfected with wild-type or deglycosylated HsPCFT fused with an HA epitope on either the N- or C-terminus. An equal amount of lysate (20 µg) was loaded and the blots were probed with a monoclonal anti-HA antibody. The numbers on the left indicate the molecular sizes in the protein ladders. Each blot is representative of three separate experiments.