FIGURE 6.

A, [³H]MTX (0. 5 μm) influx and inhibition by 1 μm folic acid at pH 5.5 and 37 °C over 1 min in R1-11 cells transiently transfected with WT-pcft and His²⁴⁷ substituted with Ala, Arg, Gln, or Glu. Data are the mean ± S.E. from three independent experiments. B, Western blot analysis of crude cell plasma membrane protein fractions from HeLa cells transiently transfected with C-HA-WT-pcft, C-HA-deglycosylated-pcft, and C-HA-tagged His²⁴⁷ Ala, Arg, Gln, and Glu substitution mutants. Equal amounts of crude cell membrane protein fractions (30 μg) were loaded, and the blots were probed with an anti-HA antibody. The numbers on the left indicate the molecular sizes in the protein ladders. β-Actin was the loading control. The blots are representative of three separate experiments.