Fig. 4. HA-Srs2 is phosphorylated during recovery from HU and its phosphorylation requires a functional Cdk1. (A and B) A log-phase culture of strain CY2735 (GAL1-SIC1) was grown in raffinose, presynchronized by αF treatment and released from the G₁ block in 0.2 M HU-containing media. Half of the culture was maintained in raffinose (A), while galactose was added to the other half of the culture to overexpress Sic1 (B). The HU block was then removed by washing the cells with YPD containing either raffinose (A) or galactose (B). Samples were taken at the time points indicated and processed for FACS or for protein extraction. Aliquots of protein extracts were analysed by western blotting performed with the 12CA5 mAb and with anti-Rad53 Ab. (C) Log-phase cultures (Log) of strain CY2735 (GAL1-SIC1) were grown in raffinose and treated with 0.2 M HU for 3 h. The culture was then divided into two parts, which were maintained in HU with (+Gal) or without (–Gal) galactose addition to induce Sic1 expression. Aliquots of protein extracts were analysed by western blotting with the 12CA5 mAb and anti-Rad53 Ab. (D) Log-phase cultures of strains CY2829 (WT) and CY2830 (cks1) were grown at 25°C and treated for 3 h with 0.02% MMS. The cultures were then shifted to 37°C in the presence of the drug. Aliquots of protein extracts were analysed by western blot analysis performed with the 12CA5 mAb and anti-Rad53 Ab.