Figure 4.

Inhibition of [³⁵S]cyclin B2-methylated UB conjugation in Δ90-arrested extracts by MAD2. Cyclin Δ90 and either MAD2 (50 μg/ml) or buffer (10 mM Tris⋅HCl, pH 7.4/10 mM NaCl) were added to interphase extract and incubated for 40 min at 23°C. The extract (20 μl) was transferred to an Eppendorf tube containing 32 μg lyophilized methylated Ub (1.6 mg/ml final concentration) and incubated for further 10 min prior to the addition of [³⁵S]cyclin B2. The [³⁵S]cyclin degradation assay was performed as described in Materials and Methods. Methylated Ub was prepared as described (29).