Role of the SAC in APC/C^Cdc20 substrate degradation. (A) Clb5 and securin degradation profiles in wild-type and mad2Δ cells. Cells are aligned using either SPB separation (left, broken line) or spindle elongation (right, solid line). Top panels show averaged and normalized traces as in Fig. 2, C and D. Bottom panels show the time of 50% substrate degradation in individual cells (Fig. S1 C). Each dot represents a single cell, and n > 90 cells per strain. For each strain, the middle bar indicates the median value, and error bars indicate the 25th and 75th percentiles. The inset in the bottom right panel shows a histogram of protein half-lives in different strains, calculated from single cell traces; n > 100 cells per strain. (B–D) Clb5 and securin degradation in nocodazole-treated cells; n > 20 cells per strain. Asynchronous cells were plated on agarose pads with 15 µg/ml nocodazole 10 min before imaging began. Representative traces from individual cells are normalized and aligned to spindle reformation (broken lines). The traces shown here were selected on the basis of two criteria: minimum overlap among traces for clarity of viewing, and inclusion of only mitotic cells, as judged by bud size. Wild-type (B and C) and mad2Δ cells (D) are shown. In B and C, representative cells with fast substrate degradation after recovery from the SAC arrest are shown in bold lines. (E) Clb5 degradation in nocodazole with CDC20 shut off; n > 20 cells. Asynchronous cells were grown in 2% galactose and plated on an agarose pad with 2% glucose and 15 µg/ml nocodazole. Representative traces began 10 min after cells were plated on the agarose pad and were selected randomly.