Figure 1

PI3K activation induces underglycosylated intracellular NIS protein expression in MCF-7 cells. (A) immunoblotting of membrane-enriched lysates from MCF-7 cells with and without tRAH treatment and MCF-7/PI3K p110α^CAAX stable clones using human NIS (hNIS) antibody. The specificity of NIS antibody was confirmed by conversion of the 86 kDa NIS band to a single 50 kDa band upon PNGase F deglycosylation (data not shown). Faint non-specific bands of 60 and 70 kDa are present. PI3K activation was assessed by total and phospho-Akt immunoblotting of total cell lysates. Actin was used as a loading control. (B) Immunoblotting of the surface protein fraction from MCF-7 cells treated with tRAH and MCF-7/PI3K p110α^CAAX stable clones using hNIS antibody. The surface fraction was isolated by cell surface biotinylation and avidin pull-down. Na⁺/K⁺ATPase was used as a loading control. (C) tRAH-treated MCF-7 cells and MCF-7/PI3K p110α^CAAX cells were labeled with hNIS antibody followed by Cy^TM3-conjugated secondary antibody (red color) and DAPI nuclear stain (blue) for immunofluorescent microscopy. Parental MCF-7 cells and secondary antibody only controls were utilized, but are not shown. Magnification = 63×.