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. 2013 Aug;27(8):3229–3238. doi: 10.1096/fj.13-229138

Figure 4.

Figure 4.

N441 in 4A NIS fully rescues protein targeting to the plasma membrane. A) Steady state I− uptake in nontransfected (Nt) and permanently expressing WT, N441/4A, or T442/4A NIS MDCK-II cells. Cells were incubated with 20 μM I− in the absence (dark bars) or presence (light bars) of 80 μM ClO4−. Results are expressed as means ± sd (pmol I−/μg DNA). Values are representative of ≥3 different experiments; in each experiment activity was analyzed in triplicate. B) Representative FACS histograms showing NIS expression under permeabilized (top panel, total) and nonpermeabilized (bottom panel, surface) conditions. Cells were stained with anti-HA Ab, followed by R-phycoerythrin-conjugated anti-rat Ab. Nontransfected cells are represented in solid gray and cells stably expressing WT, N441/4A, or T442/4A are indicated in blue, green, and red, respectively. C) Initial rates of I− uptake were determined at the indicated concentrations of I−. Calculated curves (smooth lines) were generated using the equation v = (Vmax*[I−])/(Km+[I−]) adjusted to consider background data obtained in nontransfected cells. Shown is a representative experiment of 3 independent experiments. D) Relative affinity of anti-human NIS-VJ1 Ab for WT NIS and NIS mutants. Permanently expressing MDCK-II cells were immunostained under permeabilized conditions with anti-human NIS-VJ1 or anti-HA Abs, followed by Alexa 488-conjugated anti-mouse or R-phycoerythrin-conjugated anti-rat Abs, respectively. The ratio of mean fluorescence intensity in the positive population of cell stained either with anti-human NIS VJ1 or anti-HA Abs was calculated for each mutant. The ratio obtained for WT NIS expressing cells was arbitrarily defined as 1. *P < 0.05 (ANOVA, Newman-Keuls test). E) Immunoblot analysis of membrane fractions from WT and nonglycosylated (TQ) NIS-transfected COS-7 cells performed with anti-human NIS Ab. Relative electrophoretic mobilities of the corresponding NIS polypeptides (AA: ∼50, A: ∼60, B: ∼100 kDa) are indicated. F) FACS histograms showing NIS expression under permeabilized conditions. Cells were stained with anti-HA or anti-human NIS-VJ1 Abs. Nontransfected cells are represented in solid gray and COS-7 cells expressing either WT or TQ NIS are indicated by blue and red lines.