Figure 3.

Localization of Dbf2 to SPBs and activation of Dbf2 kinase activity occur concomitantly. Cells carrying a DBF2-MYC tag (A1931) were arrested in G1 with α-factor (5 μg/ml) followed by release into medium lacking pheromone. The amount of Dbf2-Myc protein (A), Dbf2 kinase activity (B), the percentage of cells carrying Dbf2 on SPBs (n = 200; D, E) and cells containing anaphase or telophase spindles (n = 200; D) were analyzed. Clb2 protein levels (A) were examined to assess cell cycle synchrony; Kar2 was used as an internal loading control in Western blots. (C) Dbf2-myc was immunoprecipitated with the use of anti-Myc antibodies and incubated with calf intestinal alkaline phosphatase (CIP) buffer alone (IP+buffer) or with 80 U of CIP (IP+CIP). An immunoprecipitation from cells without a DBF2-MYC fusion is shown in the lane labeled “no tag”.