Fig. 2. Characterization of expression and activity of Q267E NIS in transfected COS-7 cells. (A) Immunoblot analysis. Membrane fractions (20 µg) were isolated from non-transfected COS-7 cells (NT) (lanes 1 and 4) or from COS-7 cells transfected either with WT rNIS (lane 2), rQ267E NIS (lane 3), WT hNIS (lane 5) or hQ267E NIS (lane 6). Membrane fractions were then electrophoresed, electrotransferred and immunoblotted with either 2 nM anti-Ct-rNIS Ab (lanes 1-3) or 4 nM anti-Ct-hNIS Ab (lanes 4-6). This is a representative immunoblot. Protein loading was standardized with anti-tubulin Ab (not shown). Expression of both NIS and tubulin was quantified in five different experiments. No statistically significant difference was found in the NIS/tubulin expression ratio of Q267E NIS when compared to WT NIS. (B) I^- transport. Assays were performed in the presence of 20 µM I^-/140 mM Na⁺ (colored bars) or 20 µM I^-/140 mM Na⁺ plus 80 µM perchlorate (white bars). Results are expressed as the percentage I^- uptake with respect to WT NIS. Values represent the average of at least five different experiments; in each experiment, activity was analyzed in triplicate or sextuplicate. (C) Time-course of I^- uptake in cells transfected with either WT rNIS (red squares) or rQ267E NIS (green triangles). Cells were incubated with 20 µM I^-/140 mM Na⁺ for the indicated times. Values obtained with non-transfected cells were subtracted. (D) Effect of increasing concentrations of I^- on I^- uptake. COS-7 cells not transfected (black circles) or transfected with either WT rNIS (red squares) or rQ267E NIS (green triangles) were assayed at steady-state (60 minutes) in the presence of the indicated I^- concentrations. I^- uptake values are the mean±s.d. Statistical significance of the data was calculated by t-test analysis using two-tailed P values. ^*P<0.05. Y-axes have been split into two scales (B-D).