FIG. 8.

Spp2/p58 coprecipitates with unphosphorylated Orp2/Orc2. The cdc25-22 strain containing two-myc-six-His-spb70⁺, orp2⁺-three-FLAG, and spp2⁺-three-HA was cultured at 25°C in EMM to mid-log phase and shifted to 36°C for 4 h to arrest at G₂. Cells were then released from G₂ arrest by shift to 25°C and harvested every 12 min. Chromatin fractions were prepared as described in Materials and Methods. (A) Cell cycle progression was monitored by septation index and mitotic index. (B) Kinetics of the appearance of MCM6, Orp2, Spp2/p58, and Spb70 in the chromatin fractions prepared from each time point were detected by Western blotting with their respective antibodies as described for Fig. 5B. (C) Coprecipitations of the unphosphorylated Orp2-FLAG with the HA-tagged Spp2/p58 were shown by Western blotting the anti-HA immunoprecipitates of Spp2 with anti-FLAG and anti-HA antibodies. A demonstration of phosphorylated and unphosphorylated Orp2 is shown next to the 0-min time point.