<p>U2OS cells were transfected with full length NM1-V5/His (<b>A</b>), NM1-V5/His lacking the second IQ motif (<b>B</b>), and NM1-V5/His with point mutation of basic amino acids within the NLS into alanines (<b>C</b>). Below the pictures are schematic representations of constructs used. Color coding is the same as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030529#pone-0030529-g002" target="_blank"><b>Fig. 2</b></a>. Cells were fixed 48 hours post transfection and labeled with anti-V5 antibody, pictures were obtained using wide-field microscope, scale bar: 10 µm (<b>D</b>) U2OS cells transiently transfected with Myo1c-V5/His show nuclear localization of the protein Picture is a single confocal plane, obtained by confocal microscope. Scale bar: 10 µm. (<b>E</b>) Nuclear and cytosolic extracts were prepared from liver of either wild type (WT) or NM1 knock-out (KO) mice. Equal amount of protein was resolved using SDS-PAGE and electro-transferred to nitrocellulose. Membrane was probed with anti-NM1, anti-Myo1c, anti hnRNP C1/C2 and GAPDH antibody. Signal was detected using LI-COR Odyssey infrared imaging system. (<b>F</b>) U2OS cells were transiently transfected with Myo1c-V5/His. 24 hours after transfection cells were treated with nocodazole or aphidicolin to stall the cells either in G2/M or in G1/S phase of cell cycle. After the release from the block cells were cultivated for another 24 hours. Samples were taken in indicated timepoints. Cells were labeled with antibody to V5 tag, patterns counted and divided into three groups according to the localization of fluorescent proteins. More than 100 cells were counted in each timepoint, expreriment was repeated twice with similar result.</p
