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Fig. 4. Analysis of the mutant SHOX R173C (C517T) with the insertion construct SHOX/C517T+NLS and the respective subcellular localization. (A) Schematic representation of the missense mutant C517T with the mutation in the NLS, and the insertion mutant SHOX/C517T+NLS where the NLS has been inserted in the mutant SHOX C517T. Nu, nuclear; Cy, cytoplasmic. (B) Fluorescent images of immunostained cells. The two upper panels show the cell nuclei stained with Hoechst dye (blue) and the two lower panels show SHOX immunostaining with rabbit anti-SHOX antibodies (green) in corresponding cells. The constructs in pcDNA4/TO were overexpressed in U2Os cells. The missense mutation is within the nuclear localization signal resulting in its cytoplasmic localization (arrows in the two left panels). Insertion of the nuclear localization signal near the mutated site restored its nuclear localization activity (arrows in the two right panels).





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