FIG. 2.
Phosphorylation of Nudel and NudE in M phase. (A) Specific modification of Nudel and NudE in M phase. Human hepatoma SMMC-7721 cells were synchronized at the indicated stages of the cell cycle prior to lysis. Nudel and NudE were immunoprecipitated from 200 μg of total proteins and were detected by immunoblotting with anti-Nudel IgY. (B) CIAP assay. After immunoprecipitation from mitotic SMMC-7721 lysate, half of the beads were treated either with 10 U of CIAP (lane 2) or with just buffer (lane 3). The arrow and arrowhead point to the fast and slow migration forms, respectively. (C) Phosphorylation of both Nudel and NudE in M phase. After transfection of HEK293T cells for 24 h, half of the cells were treated with nocodazole for an additional 12 h (lanes 1, 3, and 5), while the rest (lanes 2, 4, and 6) were maintained as unsynchronized controls. Cell lysates were subjected to immunoblotting with anti-FLAG antibody.