Fig. 1

Rap1 is hyper-phosphorylated in meiosis. a Schematic diagram of meiotic culture synchronization using homozygous diploid cells carrying the temperature-sensitive pat1-114 mutation and the mat-Pc cassette. b Distribution graph of the number of nuclei in meiocytes through meiosis (top left) and images of DAPI-stained cells from indicated fractions (bottom left). FACS analysis shows DNA duplication from 2C to 4C (right). c,d Western blot analysis of Rap1-3xPK from mitotic cycling cells (mit), G1 arrested cells (time 0) and meiotic cell fractions at indicated times. Anti-Cdc2 (CDK) and anti-Cdc13 (Cyclin B) antibodies were used as a loading control and meiosis synchronicity marker, respectively. c Separation of cell extracts on a standard gradient gel. d Separation of phosphorylated Rap1-3xPK on a Phos-tag gel. e Treatment of Rap1-3xPK from the 4.5 hr fraction with lambda-phosphatase and/or phosphatase inhibitors as a control. Note that fast-migrating bands of Rap1 are observed in this case due to the presence of endogenous phosphatases