CSF extract was supplemented with mRNA encoding Flag-Cdc20 that was either wild-type or mutated to serine at Thr64, Thr68 and Thr79 (3TS). After expression, calcium was added, samples were taken at the indicated time points and resolved by either conventional or Phos-tagTM SDS-PAGE. Samples were immunoblotted for the Flag-tag, Cdc27 and cyclin B2. 