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Fig. 4. Cyclin B2 is located in the nucleus and degrades in prometaphase. (A) Triple-labeling of CycB2-HA-expressing cells with antibodies against the HA-epitope (red) and {alpha}-tubulin (green); chromatin is stained with DAPI (blue). A representative cell is shown in interphase (Int), preprophase (PPB), pro-metaphase (PM), metaphase (Met), anaphase (Ana) and telophase (Tel). (B) CycB2-GFP fluorescence and the corresponding DIC images in cultured cells. Arrows in the top images indicate metaphases, and in the bottom images a cell after cytokinesis. (C) CycB2-HA is absent in stationary-phase cells and becomes detectable before S phase. Stationary phase cells from line CycB2-HA-2 were diluted with fresh medium and immediately incubated with BrdU labeling reagent. Aliquots were taken at 3 hour intervals and the initiation of S phase was determined by immunostaining for BrdU incorporation using an antibody against BrdU. The percentage of cells with CycB2-HA protein was determined by immunostaining using an antibody against the HA-epitope. Scale bars: 10 µm.