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. 2011 Oct 14;286(49):42758–42769. doi: 10.1074/jbc.M111.228411

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© 2011 by The American Society for Biochemistry and Molecular Biology, Inc.

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FIGURE 1. — Subnuclear distribution of CENP-W. A, localization of EGFP-tagged CENP-W in HeLa cells. After EGFP-CENP-W was transfected to HeLa cells, the green fluorescence images were captured using fluorescence microscopy at 200× magnification. As nucleolar marker proteins, B23 and fibrillarin were conjugated with RFP and co-transfected with CENP-W. DAPI signals were also applied to localize the nucleus of each cell. B, localization of EGFP-CENP-W in SKOV-3 cells. C, localization of FLAG-CENP-W in HeLa cells. After HeLa cells were transfected with the FLAG-tagged CENP-W, CENP-W was visualized using anti-FLAG and FITC-labeled anti-mouse secondary antibody and B23 using anti-B23 and Cy3-labeled anti-rabbit antibody. D, localization of FLAG-CENP-W in time course. After transfection of FLAG-CENP-W in HeLa cells, the distribution of CENP-W was analyzed at different time points using immunofluorescence microscopy. The endogenous B23 (red) was also stained at 48 h. E, localization of CENP-W in A549 stable cells. After extraction of soluble proteins with 0.5% Triton X-100, cells were immunostained with anti-FLAG antibody. F, nucleolar fractionation of CENP-W-expressing cells. The nucleolus fraction was obtained from either transiently expressed 293T cells or A549-FLAG-CENP-W stable cells. Each fraction was analyzed by Western blotting with anti-FLAG, anti-α-tubulin, and anti-B23 antibodies. T, total cell extract; C, cytoplasmic fraction; N, nuclear fraction; Np, nucleoplasmic fraction; No, nucleolar fraction.