Fig. 2. HA-Srs2 phosphorylation requires checkpoint activation and entry into S phase. (A and B) Aliquots of protein extracts, prepared from CY2715 cells taken at the indicated time after α-factor (αF) release in the absence (A) or presence (B) of 0.02% MMS, were analysed by western blotting with 12CA5 mAb, anti-Rad53 Ab and the 6D2 mAb recognizing the pol–prim B subunit. Aliquots of cells taken at the indicated time were also processed for FACS analysis as described in Materials and methods. (C and D) CY2715 cells were blocked in G₁ by αF treatment, as described in Materials and methods. G₁-arrested cells were treated for 15 min with 0.25 µg/ml 4-NQO; the drug was then removed by washing the cells with YPD containing (C) or not containing (D) αF to maintain the G₁ block or to allow cell cycle progression. At the indicated times after 4-NQO removal, aliquots of protein extracts were analysed by western blotting with 12CA5 mAb and anti-Rad53 Ab. At the same time points, cell samples were also processed for FACS analysis.