Figure 2.—

Dsn1–Ser264, a Cdk1 target site, is hypophosphorylated prior to anaphase. (A) A polyclonal phospho-specific antibody was custom-made by Pacific Immunology against phospho-Ser264 Dsn1 using peptide GGSTII(pS)PHKDIPEED. Dsn1–WT–FLAG (SBY7902) and Dsn1–S264A–FLAG (SBY8123) were immunoprecipitated using anti-FLAG antibodies as previously described (Akiyoshi et al. 2009), immunoblotted using the phospho-specific antibody at a 1:5000 dilution, and then reprobed with monoclonal FLAG antibodies. (B) Ser264 phosphorylation depends on Cdk1/Cdc28. Dsn1–FLAG proteins immunoprecipitated from cdc28–as1 mutant cells (SBY8042) (Bishop et al. 2000), treated with DMSO or 10 μm 1NM-PP1 for 30 min, were analyzed as in A. (C) Dsn1–FLAG proteins immunoprecipitated from asynchronous or preanaphase cells that were treated with 15 μg/ml nocodazole for 3 hr were analyzed as in A. (D) pGAL–CDC20 cells expressing Dsn1–FLAG (SBY8008) were asynchronously grown in galactose media and arrested in metaphase by adding glucose for 3 hr to deplete Cdc20. Dsn1–FLAG was immunoprecipitated and analyzed as in A.