Fig. 4. Cdc8 is acetylated in vivo. (A,B) cdc8-110 cells expressing additional integrant Tm genes under the control of the nmt41 promoter were grown at 36°C in EMM2 lacking thiamine. (A) The percentage of cells with two (black bars) or more than two (grey bars) nuclei were scored for each strain after 4 hours. (B) Growth curves generated for each strain over a 6 hour growth at 36°C. Only Cdc8 and smooth muscle Tm were capable of fully complementing the cdc8-110 mutation. Endogenous Cdc8 was purified from mid-log phase S. pombe cells and analysed alongside total protein extracts by silver staining of SDS-PAGE gels (C) and western blot analysis using Cdc8 antisera (D). A single doublet migrating at 30kDa was revealed by silver staining (C), while Cdc8 antisera recognised identical doublets in each lane. (E) Mass spectroscopy analysis of the purified endogenous Cdc8 revealed 20% of this protein had a mass of 18964.1 (predicted mass of Cdc8: 18,964.7 Da), while the remaining 80% had a mass pf 19005.5 Da, which corresponds to the predicted mass of acetylated Cdc8.