FIG. 2.

pRB, but not p130 nor p107 phosphorylation status is modulated during the cell cycle of 293 cells. (A) 293 cells were incubated with nocodazole for 18 h. Loosely attached cells were detached by shaking the tissue culture dishes and then collected, washed, and placed in fresh medium. Cells were then harvested at the indicated time points. The phosphorylation status of the three pocket proteins was assessed by Western blot analysis by using antibodies to p130, p107, and pRB (three upper panels). The kinase activity associated with cyclin A was also determined from the same whole protein extracts (lower panel) as in Fig. 1B. (B) 293 cells were incubated with hydroxyurea for 16 h and then washed and incubated with fresh medium. The phosphorylation status of the three pocket proteins was determined by Western blot analysis as in panel A. The cell cycle distribution of cells collected at each time point was determined by FACS (both panels). The percentage of cells at each stage of the cell cycle is indicated below. The differently phosphorylated forms of each pocket protein are indicated.