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Determination of MTX influx under trans-stimulation conditions in R1-11 cells transiently transfected with mock, WT-PCFT, or PCFT mutants. Influx of 0.5 μM [3H]MTX at pH 5.5 was assessed in control cells and cells preloaded with nonlabeled 200 μM MTX for 20 min at 37°C.