Fig. 6. 53BP1 is hyperphosphorylated in mitosis. 53BP1 was immunoprecipitated from protein extracts prepared from asynchronous (A, lane 1), mitotic (M, lane 2 and 3), and colcemid blocked (M+C, lane 4,5) HeLa cells. The precipitates were incubated in the presence (lanes 3,5) or in the absence (lanes 1,2,4) of protein phosphatase (PPase). The proteins were run on a gel and the electrophoretic mobility of 53BP1 was examined by western blotting. In mitotic cells (lane 2), 53BP1 exhibited a lower electrophoretic mobility compared with that in interphase cells (lane 1). The retardation was even more pronounced in mitotically blocked cells with colcemid (compare lane 2 versus 4). The 53BP1 electrophoretic retardation was eliminated by the protein phosphatase treatment (lanes 3,5). (B) Mitotically blocked HeLa cells exhibits a strong crescent shaped 53BP1 signal on kinetochores. Colcemid treated HeLa cells were double-stained for 53BP1 (green) and CENP-E (red) as in Fig. 2, bottom row. Inset in panels b, c, and d are a magnified view of the kinetochore pair shown by the arrow. The identity of the signal(s) displayed in each panel is written above the pictures. DNA stained with DAPI is shown in blue (a). Images were recorded with a Delta Vision microscope.