Fig. 3. Bfa1 is a phosphoprotein. (A) Mid-log cells of strain SLY105 (3HA-BFA1) were harvested or arrested by using -factor (F), HU or nocodazole (Noco) for 3 hours before harvesting. Following protein extraction, an immunoblot was prepared and probed with 12CA5. The Roman numerals on the right indicate the various Bfa1 species. (B) Mid-log cells of strain SLY105 were harvested immediately or following treatment with nocodazole for 3 hours. Protein extraction was performed as usual or in lysis buffer without phosphatase inhibitors (+/- PI). 70 µg extract was incubated with calf alkaline phosphatase (CAP) at 30°C for 30 minutes prior to SDS-PAGE and immunoblotting. (C) Phosphorylation of Bfa1 is dependent upon Bub2. Strain SLY111, isogenic to SLY105 but containing bub2, was treated as described in (A).