Figure 3.

All phases of mitosis show low levels of endogenous Mcm10. (A) and (B) For evaluating subcellular localization of endogenous proteins, HeLa cells were fixed in formaldehyde, permeabilized with Triton X-100 and DNA was stained with 4, 6-diamidino-2-phenylindole (DAPI). To visualize Mcm10, immunofluorescence was performed using a rabbit polyclonal anti-Mcm10 antibody in combination with an anti-rabbit TRITC secondary antibody while microtubules were visualized with a mouse monoclonal antibody to alpha-tubulin conjugated to FITC. From unperturbed asynchronously growing culture, HeLa cells that are in prophase, metaphase, anaphase and telophase were identified and have been marked by arrowheads in panel (A). The arrangement of immunofluorescence images obtained from a single field has been illustrated in panel (B) and the bottom right square of each field is a merge of DAPI, FITC and TRITC images. Field numbers have been marked at the top-right corner of each field. (C) To obtain cells arrested in prometaphase (shown by arrowheads), HeLa cells were treated with colchicine, nocodazole, taxol or vincristine as described in Figure 1 and evaluated for Mcm10 levels by immunofluorescence.