Fig. 2. Location of protein B23-GFP and mutants during interphase and mitosis. (A) Localization of GFP-tagged wild-type and endogenous protein B23 during different phases of mitosis. Both the proteins showed similar localization during various phases of mitosis. Upper panels, GFP-tagged expressed protein; lower panels, endogenous protein as detected by immunofluorescence using a monoclonal antibody. Arrows indicate the position of NDF in an anaphase cell and arrowheads indicate the position of PNBs in the newly forming nucleus at telophase. (B) Expression of the wild type protein and the mutants tagged with GFP. Immunoblot was developed with anti-GFP mAb. B23-GFP (lane 1), S125A-GFP (lane 2), S125E-GFP (lane 3) and untransfected cell lysate (lane 4). (C) Location of (a) wild-type B23-GFP, (b) S125A-GFP, (c) S125E-GFP and (d) endogenous B23 detected by immunofluorescence using a monoclonal antibody in interphase HeLa cells. All the fusion proteins localized to the nucleolus as did the endogenous B23 protein. Corresponding images of Hoechst 33342-stained DNA are shown in (a'), (b'), (c') and (d'). Bars, 10 µm.