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Fig. 7. Localization of the 3F3/2 phosphoepitope and checkpoint proteins in cells expressing N-terminal fragments of MLCK. Scale bars, 10 µm. (A) Immunostaining with the 3F3/2 antibody was used to examine the presence of unattached or mono-oriented chromosomes in transfected cells (left). DNA was visualized with DAPI (right). In cells expressing the IgG domain or IgG domain plus five DXRs, 3F3/2 staining was observed in more than 50% of cells with a visible metaphase plate, typically on misaligned/uncongressed chromosomes. In untransfected metaphase cells or cells expressing GFP or the five DXRs, 3F3/2 fluorescence was not detected. In transfected cells, the Bub3 (B) and Mad1 (C) checkpoint proteins (top) localized to unattached kinetochores during prophase and prometaphase (a,c,e). In cells expressing the IgG domain or IgG domain plus five DXRs, bright Bub3 and Mad1 staining was observed on the kinetochores of misaligned chromosomes near spindle poles or uncongressed chromosomes in metaphase cells (arrows), whereas only residual staining was detected on the kinetochores of aligned chromosomes. Chromosomes and nuclei were stained with DAPI (middle). Merged images of checkpoint proteins (red) and DNA (blue) (bottom).
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