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. 2006 Feb;17(2):814–823. doi: 10.1091/mbc.E05-08-0729

Figure 2.

Figure 2.

Defective Brd4 reloading in Brd4+/- cells. (A) Reduced Brd4 levels in Brd4+/- clones. Immunoblot analysis was performed with the whole cell lysates (30 μg) from parental ES cells (WT) and three representative Brd4+/- clones (C12, C16, and C27) for Brd4 and α-tubulin as a loading control. (B) Brd4+/+ and +/- cells were treated with nocodazole for 8 h; mitotic cells were incubated in fresh media for 40 min and immunostained for Brd4 localization. Left, an example of a Brd4+/+ cell in which Brd4 was fully reloaded on the segregating chromosomes. α-Tubulin was stained to visualize mitotic spindles. Right, an example of a Brd4+/- cell in which Brd4 remained outside of the segregating chromosomes. Bar, 5 μm. (C) Brd4 reloading was monitored by immunostaining of parental Brd4+/+ and three +/- clones that had been treated with nocodazole and released in fresh media for 40 min as described above. The black columns represent the percentage of cells in which Brd4 was reloaded on chromosomes, and the white columns represent those in which Brd4 was not reloaded on chromosomes. A representative of three independent tests is shown. More than 200 cells with segregating chromosomes (anaphase/telophase cells) were examined in each experiment.