Figure 8.

Interaction of mutant Cdc6p with Cdc28p. Strain BJ2168 was transformed with plasmids pELS71 (wild type), pELS72 (Δ3–16), pELS73 (Δ13–26), pELS74 (Δ23–26), and pELS75 (Δ33–46). Cells were grown overnight in synthetic medium prepared with raffinose and lacking leucine. Expression of Cdc6 proteins was induced for 4 h with 2% galactose. Extracts were prepared, and immunoprecipitations were carried out as described in MATERIALS AND METHODS. Top panel, 10 μg of protein was electrophoresed on a 10% gel, blotted to a polyvinylidene difluoride membrane (Millipore), and probed with anti-myc monoclonal antibody 9E10. The apparent molecular mass of Cdc6-Myc9p is 120 kDa. Bottom two panels, 9E10 immunoprecipitates were electrophoresed on a 12.5% gel and blotted to a polyvinylidene difluoride membrane. The top half of the membrane was probed with anti-myc monoclonal antibody 9E10 (middle panel), and the bottom half of the membrane (bottom panel) was probed with anti-Cdc28p polyclonal antibody RAA1. Protein blots were developed with ECL reagents (Amersham). The lane on the left labeled C is a control for the Cdc28p antibody reaction in which extract was run on the gel without previous immunoprecipitation.