Fig. 1. Bfa1 and Bub2 physically associate. (A) Bfa1 negatively regulates Dbf2 kinase activation. Isogenic strains carrying integrated DBF2-6MYC, YDF20 (apc2-8) and SLY103 (apc2-8 bfa1) were grown to mid-log phase at 25°C and arrested with -factor (F) or with nocodazole at 37°C (Noco at 37°C) or at 37°C (37°C) for 3 hours. Extracts were immunoprecipitated with anti-Myc (9E10) and Dbf2 kinase assayed (lower panel). The upper part of the kinase assay gel was immunoblotted and probed with 9E10 antibody to control for loading. (B) Bfa1 and Bub2 associate across the cell cycle. Strain SLY106 (BUB2-13MYC 3HA-BFA1) was grown to mid-log and synchronised with -factor. Cells were sampled for protein extraction at the times shown. 300 µg extract was immunoprecipitated using anti-HA (12CA5) antibody, analysed using SDS-PAGE, immunoblotted and probed with 9E10. Lanes 1-3 show an untagged control (CG378) and two singly tagged strains. The Ig band serves as a loading control. The bottom panel shows budding in the synchronised culture.