Fig. 2.

Analysis of charged residues within IL-2 of NIS. (A) Sequence alignment of IL-2 of SLC5A family members. The R124 position in NIS and corresponding residues in other members are indicated. Positively charged residues are indicated in blue and negatively charged ones in red. Left and right shaded sequences represent parts of TMS III and IV, respectively, according to the crystal structure of vSGLT. (B) Flow cytometry of non-transfected (NT) COS-7 cells or COS-7 cells transfected with WT or IL-2 NIS mutants under non-permeabilized conditions to assess NIS expression at the plasma membrane (PM). Staining was done using anti-NIS VJ1 Ab, followed by Alexa-488-conjugated anti-mouse Ab. (C) Steady-state I⁻ transport in WT or R111A, E119A, E122A, R124A and R127A NIS-transfected COS-7 cells. Cells were incubated with 20 µM I⁻ in the absence or presence of 80 µM ClO₄⁻. Values are expressed in pmol I⁻/µg of DNA ± s.d. and are representative of three different experiments; each experiment was performed in triplicate.