Fig. 3. Securin and cyclin B1 have a similar degradation pattern in G₁. (A) Cells were stably transfected with the securin–CAT expression vector which constitutively transcribes a reporter fusion protein between the N-terminal 87 residues of human securin and a bacterial CAT protein. Cells were synchronized as described and assayed for CAT activity. The CAT activity was calculated as the ratio of the amount of diacetylated [¹⁴C]chloramphenicol to that of total [¹⁴C]chloramphenicol (diacetylated + non-acetylated) as quantified by a phosphoimager. The changes in the activities of securin–CAT (open circles) and cyclin B1–CAT (filled squares) (shown in Figure 1) were plotted as a function of the cell cycle phases when released from a nocodazole block. (B) Cells stably expressing CAT (filled circles), cyclin B1–CAT (filled squares) and securin–CAT (open circles) were arrested by serum deprivation in G₀ and released by serum stimulation. Cells were harvested and analyzed for CAT activity at the indicated time points. (C) Cells were synchronized at prometaphase by nocodazole arrest and mitotic shake-off, released into fresh medium and harvested at the indicated time points for immunoblotting with securin, cyclin B1 and cdk1 antibodies. (D) Prometaphase-arrested cells were washed and released into fresh medium for 2 h, and then either harvested or grown for an additional 2 h with or without ALLN. Cell extracts were immunoblotted with securin or cdk1 antibodies. Cdk1 is constant in G₁ and served as a loading control.