Supplemental Material

This article contains the following supporting material:

• Figure 10 Supplemental - Consensus CDK phosphorylation sites of Cdc6 are essential for the association of Cdc6 with Cdc28.
  Cells of strains YSH63 (GAL-CDC6*-9XMYC) and YSH61 (GAL-CDC6-9XMYC) were grown to log phase in medium containing YEP and raffinose (2%). Galactose (1%) was added to induce CDC6* or CDC6 expression. 40min later, cells were harvested and lysed in yeast lysis buffer (50mM Tris pH 7.5, 250mM NaCl, 50mM NaF, 5mM EDTA, 0.5% Triton X-100, PMSF, Leupeptin, Pepstatin, TPCK, and STI) (Honey et al. , 2001). Cdc6*-9XMYC and Cdc6-9XMYC were immunoprecipitated from clarified cell lysates (equivalent to 4mg total protein) using 9E10 (anti-myc) monoclonal antibody on Protein-G beads. Immunoprecipitates were washed four times and then analyzed by immunoblotting for Cdc28 using anti-Cdc28 (Cdc28-181, BF, unpublished) antibody.