Fig. 6: Esc2, STUbL, and Cullin8 mutants have delayed DNA damage checkpoint activation.

Kinetics of Rad9 and Rad53 phosphorylation in WT, esc2Δ, slx5Δ, and cul8Δ cells. Rad9-FLAG-expressing cells were synchronized in G1 phase with α-factor and then released in YPD medium containing 0.033% MMS. Samples were taken at the indicated timepoints and prepared for western blot analysis using anti-Rad53 and anti-FLAG antibodies. Immunodetection of Tubulin served as a loading control. Checkpoint-deficient ddc1Δ cells were used as control. Three independent experiments (n = 3) were used for the quantification of Rad9 and Rad53 phosphorylation; the bars represent the mean values ± SEM. P values were obtained by using a two-tailed unpaired Student’s t test with Welch’s correction, the asterisks indicate *P < 0.05, **P < 0.01 and ***P < 0.001. P values for Rad9 quantification: esc2Δ = 0.0302 (45 min); slx5Δ = 0.0479 (30 min), 00336 (45 min); cul8Δ = 0.0344 (30 min), 0.0206 (45 min); ddc1Δ = 0.0259 (30 min), 0.0092 (45 min). P values for Rad53 quantification: esc2Δ = 0.007 (30 min), 0.0264 (45 min); slx5Δ = 0.0343 (30 min), 00392 (45 min); cul8Δ = 0.0283 (30 min), 0.0346 (45 min); ddc1Δ = 0.0005 (30 min), 0.0004 (45 min). NS stands for not significant. Source data are provided as a Source data file.