NPM, a shuttling protein, is mainly localized in the nucleolus. (Top) Mechanism of nucleocytoplasmic traffic of NPM wild-type (NPMwt). Green boxes indicate NPMwt protein; red circles, tryptophan residues; NES motif, nuclear export signal motif; and NLS, nuclear localization signal. The nuclear import of NPM (arrows) greatly predominates over the nuclear export (dotted arrow). Thus, NPM is mainly localized in the nucleolus. (Middle) Confocal analysis of NIH-3T3 cells expressing enhanced green fluorescent protein (eGFP)–NPMwt fusion protein. The bulk of NPMwt (green) colocalizes in nucleoli with C23/nucleolin (red), as revealed using a specific antibody. Images were collected with a Zeiss LSM 510 confocal microscope (Zeiss, Jena, Germany) using a Plan Apochromat 100×/1.4 NA oil objective and the LSM 5 software (Zeiss) for image acquisition. An Alexa 543-conjugated secondary antibody (Molecular Probes, Eugene, OR) was used for C23/nucleolin staining; nuclei were stained with To-Pro3 (Molecular Probes). Three-dimensional reconstruction of the confocal images and electronic cuts of nucleus and nucleoli to analyze the subcellular distribution of NPMwt (green) and C23 (red) proteins were performed with Imaris software (Bitplane, Zurich, Switzerland).