Figure 6.

N-terminal hCdc20 and hCdh1 bind substrates directly. (A) ³⁵S-labeled in vitro translated Xkid, geminin, and securin, each isolated by gel filtration over a Superdex 200 column incubated in the presence of Nickel beads, or bacterially expressed H6Cdc20N120 or H6Cdh1N125 bound to nickel beads. (B) Pull-down assays using bacterially expressed H6Cdc20N120 or H6Cdh1N125 cross-linked to Pierce UltraLink beads incubated with bacterially expressed and purified human H6-securin, baculovirus-expressed and -purified mB99, or bacterially expressed and purified Xenopus H6-Ubcx. Visualization of his-tagged proteins was achieved following Western transfer and blotting with a QIAGEN α-pentahis antibody. Five percent input is shown for H6securin, and 10% input is shown for H6-mB99 and H6-Ubcx.