Figure 1.

UbcH7 is regulated in a cell cycle–dependent manner. (A) Cell cycle profile of cells synchronized by treatment with 2 mM HU. HeLa cells were treated for 18 h, HU-containing medium was washed out, and cells were allowed to resume cycling in medium without drugs. Mean fluorescence intensity relating to DNA content is plotted on the X axis, and cell number is plotted on the Y axis. (B) UbcH7 levels are low in S phase and rise in G2/M. HeLa (left panel) or HLE (right panel) cells were treated with HU for 18 h and then released from drug treatment to resume cycle. Lysates, prepared from samples at times after drug release, were blotted using α-UbcH7, α-UbcH10, α-Ubc3, α-Ubc2, and α-E1, the latter being used as a loading control. We and others have demonstrated no alterations in E1 levels during different phases of the cell cycle (Stephen et al., 1996; Liu et al., 2004). Because E2 levels from the same samples were assessed on different immunoblots, E1 controls are shown for each blot. The majority of cells (≥75%) were in the cell cycle phase indicated. Bottom, quantitation of gels shown above. (C) UbcH7 is high in mitotic cells. Immunofluorescent staining with α-UbcH7 (left), α-tubulin (middle), or isotype matched control antibody (right) on cells treated for 18 h with nocodazole. Mitotic cells are noted with a white arrow in all panels.