Expression and plasma membrane targeting of NIS mutants. (A) RT-PCR. HepG2 cells were cultured in 25-cm2 flasks and transfected with wild-type (wt), R9A, E79A, R82A, K86A, D163A, H226A, R228A, D233A, D237A, R239A, R241A, D311A, D322A, or D331A plasmid DNAs. Total RNAs were extracted and 1 μg of total RNA was reverse transcribed. The resulting cDNAs were then amplified by PCR. PCR products were resolved in 1% agarose gels and visualized with ethidium bromide. (B) Western blot. HepG2 cells were cultured in 25-cm2 flasks and transfected with wt or mutated NIS DNAs. The NIS and β-actin proteins in the cellular lysates were detected by Western blot. (C) Immunofluorescent staining. HepG2 cells were cultured on glass coverslips and transfected without (blank) or with pcDNA3.1 (mock), wt, or mutated NIS DNAs for 2 days. Cells were then treated with anti-NIS antibody, stained with fluorescence-conjugated secondary antibody, and evaluated under a confocal microscope. Magnification, 400×. Similar results were obtained in three independent experiments.