Fig. 3. flp1p is phosphorylated during mitosis. The strain cdc25-22 flp1-HAc was synchronised by arrest-release and protein samples were prepared from cells at the indicated times. Note that a slower-migrating form of flp1p-HA appears during mitosis. The black arrowhead (B) indicates the rapidly migrating, interphase form of the protein, the grey arrowhead indicates the form present in G₂ cells. (A) The progression through mitosis after release from the cdc25-22 block, as determined by fixing cells at each time point, and staining with DAPI and Calcofluor. (B) Western blot of protein samples prepared at the indicated times, probed with 12CA5 and TAT-1, to reveal flp1p-HA and -tubulin, respectively. (C) flp1p-HA was immunoprecipitated from protein samples prepared at the indicated times. After washing, the 45 minute immunoprecipitate was divided in half and treated with alkaline phosphatase (+), or not (-). A western blot of these protein samples was probed with 12CA5 to reveal the position of flp1p-HA.