Fig. S5.

CCT complex releases Mad2–Cdc20 subcomplex from MCC. MCC disassembly by CCT5 complex at the indicated concentrations was carried out as described in Materials and Methods. Released material was passed through 0.45-μM centrifugal filters and subjected to immunoprecipitation with anti-Cdc20 beads by rotation at 4 °C for 2 h. The beads were then washed three times with a buffer consisting of 50 mM Tris⋅HCl (pH 7.2), 20% (vol/vol) glycerol, 300 mM NaCl, and 1% (vol/vol) Nonidet P-40. Immunoprecipitates were resolved by SDS/PAGE and immunoblotted for Myc–Cdc20 and Mad2. Where indicated, additions were as described in Fig. 3B. Arrows on the Right indicate the electrophoretic migration of marker proteins (in kilodaltons).