Figure 2: Cyclin A2–Cdk2 phosphorylates Cdc20.
From: Interphase APC/C–Cdc20 inhibition by cyclin A2–Cdk2 ensures efficient mitotic entry
(a) Western blot of input and Cdc20-purified samples from HeLa cells treated with cyclin A2 siRNA (CycA2#1-2), synchronized with thymidine and collected 8 h after release from the thymidine block. Cdk1 inhibitor RO3306 was added 2 h before cells were collected. (b,c) Western blot of input and Cdc20-purified samples from HeLa cells synchronized with thymidine released for 6 h and then treated for 2 h with either Cdk1/2 Inhibitor III or RO3306. (d–f) Western blot of input and Cdc20-purified samples from HeLa cells synchronized with thymidine released for 6 h and then treated for 2 h with okadaic acid (OA). (g) HeLa cells were synchronized with a double thymidine block and depleted of PP1α, PP1γ or PP2A catalytic and scaffold subunit (PP2A). Six or nine hours after release from the second thymidine block, cells were collected. (h) Western blot of purified Cdc20 from experiment as in g. (i) Quantification of the signal obtained from the phospho-specific antibodies normalized to the pan Cdc20 signal. The phosphorylation level 9 h after thymidine release was set to 1. (a–i) are representative of at least two independent experiments.
