Figure 6.

 Phosphorylation of Mcm2 at the G₁-to-S-phase transition is blocked when Cdc7–Dbf4 kinase is inactivated. The isoform distributions of Mcm2 (left) and Mcm5 (right) were analyzed by immunoblots of two-dimensional protein gels with monospecific antibodies. dbf4-1 cells used in this assay were first arrested at Start by α factor at 30°C. Proteins were isolated from cells released from the α factor block and then arrested at G₁-to-S-phase transition by shifting to 37°C (top) and from cells released from the α factor block and then arrested at early S by adding HU to the culture (bottom). In the first dimension gel, proteins are separated by isoelectrofocusing (IEF). In the second dimension gel, proteins are separated by molecular weight (MW). The two-dimensional gels were aligned with chemiluminescence signals from protein aggregates that did not enter the first dimension gel (Young and Tye 1997).