Figure 7.

Lag2 dose dependently inhibits the binding of Cdc34 to SCF^Cdc4 complex in vitro. (A) The indicated recombinant proteins were mixed and incubated on ice for 1 h. Binding of Cdc34 and SCF^Cdc4 complex was detected by GST pull-down analysis and followed immunoblot analysis (IB) with anti-Cdc53 and anti-GST. (B) Ni²⁺-agarose beads containing Cdc53/His₆-Rbx1 complex was mixed with the indicated amount of His₆-Lag2 at 4°C for 1 h. After washing, bound proteins were separated by SDS/PAGE and subjected to immunoblot analysis (IB) with anti-Cdc53 and anti-His.