Figure 4. Role of phosphorylation by... | Rockefeller University Press

Figure 4.

$Figure 4. Role of phosphorylation by Cdk1 in APC/CCdc20 substrate degradation. (A) Degradation profiles of GFP-tagged securin-2A, wild-type securin, and Clb5, as in Fig. 3 A. n > 70 cells per strain, and in the inset, n > 160 cells per strain. (B) Fraction of securin or securin-2A remaining when spindle elongation occurs. Single-cell traces of GFP were smoothed, and the fraction remaining was calculated as the GFP intensity at spindle elongation divided by maximum GFP intensity. Each dot represents a single cell (n > 100 cells per strain). For each strain, the middle bar indicates the median value and error bars indicate the 25th and 75th percentiles. (C) Dbf4 ubiquitination by APC/CCdc20 in vitro. Radiolabeled Dbf4 N-terminal fragment (residues 1–236) was produced by in vitro translation and incubated with buffer, purified Clb2-Cdk1, or both Clb2-Cdk1 and Cdc14, before the addition of purified APC/C, Cdc20, and other ubiquitination components for the indicated times. Reaction products were separated by SDS-PAGE and analyzed by autoradiography. (D) Dbf4 and Dbf4-A ubiquitination by APC/CCdc20 or APC/CCdh1 in vitro, as in C. (E) Degradation profiles of Dbf4-A-GFP and wild-type Dbf4-GFP, as in Fig. 3 A; n > 70 cells per strain.$

Role of phosphorylation by Cdk1 in APC/C^Cdc20 substrate degradation. (A) Degradation profiles of GFP-tagged securin-2A, wild-type securin, and Clb5, as in Fig. 3 A. n > 70 cells per strain, and in the inset, n > 160 cells per strain. (B) Fraction of securin or securin-2A remaining when spindle elongation occurs. Single-cell traces of GFP were smoothed, and the fraction remaining was calculated as the GFP intensity at spindle elongation divided by maximum GFP intensity. Each dot represents a single cell (n > 100 cells per strain). For each strain, the middle bar indicates the median value and error bars indicate the 25th and 75th percentiles. (C) Dbf4 ubiquitination by APC/C^Cdc20 in vitro. Radiolabeled Dbf4 N-terminal fragment (residues 1–236) was produced by in vitro translation and incubated with buffer, purified Clb2-Cdk1, or both Clb2-Cdk1 and Cdc14, before the addition of purified APC/C, Cdc20, and other ubiquitination components for the indicated times. Reaction products were separated by SDS-PAGE and analyzed by autoradiography. (D) Dbf4 and Dbf4-A ubiquitination by APC/C^Cdc20 or APC/C^Cdh1 in vitro, as in C. (E) Degradation profiles of Dbf4-A-GFP and wild-type Dbf4-GFP, as in Fig. 3 A; n > 70 cells per strain.