Fig. 2.
Characterization of hPCFT protein expression, membrane localization, and pH-dependent transport in R2/hPCFT4 and R2/VC cells. A, Western blot analysis of hPCFTMyc-His6 in R2/hPCFT4 and R2/VC cells. Membrane fractions (10 μg) were analyzed by SDS-polyacrylamide gel electrophoresis and immunoblotting with myc monoclonal antibody. B, immunofluorescence of R2/hPCFT4 and R2/VC cells. Cells were fixed with paraformaldehyde, permeabilized with Triton X-100, stained with anti-myc antibody, and visualized using confocal microscopy. C, hPCFT transport activity in R2/hPCFT4 and R2/VC cells was assessed by measuring uptake of 0.5 μM [3H]Mtx at 37°C for 2 min at pH 5.5, 6.0, or 6.5 in MES-buffered saline and at pH 6.8 or 7.2 in HEPES-buffered saline. Internalized [3H]Mtx was normalized to total protein and expressed as a percentage of the transport activity at pH 5.5. D, hRFC transport activity in PC43-10 and R2 cells was determined by measuring uptake of 0.5 μM [3H]Mtx at 37°C for 2 min at pH 5.5, 6.0, or 6.5 in MES-buffered saline and at pH 6.8 or 7.2 in HEPES-buffered saline. As above, internalized [3H]Mtx was normalized to total cellular protein and expressed as percentage transport at pH 7.2. Transport results are presented as mean values ± S.E. from six experiments.