Fig. 1. Fzr binding to the APC/C is associated with its G₁-specific activity. (A) NIH 3T3 cells stably expressing cyclin B1–CAT were synchronized at prometaphase with nocodazole and subsequent shake-off of the rounded cells. They were washed and released into fresh medium and harvested at the indicated time points. Cells synchronized by this method pass the G₁–S transition at ∼7–9 h. Cell extracts were immunoprecipitated with anti-cdc27 beads. The immunoprecipitates were analyzed by immunoblotting with fzr and cdc27 antibodies. (B) The cells were analyzed further for CAT activity, an indication of the stability of the cyclin B1–CAT and thus of APC/C activity or inactivity. (C) Serum-starved, G₀-arrested fibroblasts were stimulated with serum to re-enter the cell cycle and were harvested at the indicated time points. They were immunoblotted with fzy antibodies and with α-actin antibodies which served as a loading control. Entry into S phase takes place at 12–15 h after stimulation. An extract of nocodazole-arrested cells, at a time when fzy levels peak in the cell, was used as a positive control.