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Fig. 4. Surface biotinylation analysis of Q267E NIS. Non-transfected COS-7 cells (NT) or COS-7 cells transfected with WT rNIS, rQ267E NIS, WT hNIS or hQ267E NIS were biotinylated with 1 mg/ml Sulfo-NHS-SS-biotin (A,D) or Sulfo-NHS-LC-biotin (B,C,E,F). Immunoblot analysis of surface biotinylated polypeptides precipitated with streptavidin-agarose beads was performed with either 2 nM anti-Ct-rNIS Ab (A) or 4 nM anti-Ct-hNIS Ab (D). This is a representative immunoblot. Nitrocellulose membranes were stripped and reprobed with anti-Na+/K+-ATPase alpha subunit antibody (not shown). Expression of both NIS and Na+/K+ ATPase alpha subunit was quantified in five different experiments. No statistically significant difference was found between the NIS/ATPase-alpha-subunit expression ratio of Q267E NIS and WT NIS. (B,E) Blot analysis of surface-biotinylated polypeptides immunoprecipitated with anti-Ct-rNIS (B) or anti-Ct-hNIS (E) and probed with streptavidin-HRP. (C,F) Stripped nitrocellulose membranes were reprobed with anti-Ct-rNIS (C) or anti-hNIS VJ1 (F).





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