Figure 1.

Phosphorylation of Cac1p in cdc7–1 and cdc28–1 mutants. (A) Cac1p-MYC18 was immunoprecipitated from wild type (W303) cell extracts. The samples were treated without (lanes 1, 2) or with lambda phosphatase (lane 3) and run on separate 6.5% SDS-50 μM PhosTag^TM-polyacrylamide gels containing or not 100 μM MnCl₂ as indicated. In the left-hand panel the cells shown above the lanes were grown at 23°C (lanes 4–7) and then shifted to 37°C for one hour (lanes 8–11) before extracts were prepared by boiling in Laemmli buffer. All samples were analyzed Western blotting with anti-MYC antibody. “- P-” indicates the mobility of the phosphorylated Cac1p-MYC. One of 3 independent experiments is shown. (B) Cells were arrested with α-factor for 3 hours at 23°C, moved to fresh YPD medium for 20 min and then split and grown for 30 min at 23°C and 37°C, respectively. Samples were taken out at the indicated times and analyzed by FACS. Cells arrested in M-phase with Nocodazole (M) show 2c content. Numbers 1–12 indicate the samples corresponding to the lanes in C. Left-pointing arrows highlight the 23°C and 37°C 30 min samples for comparison. (C) Samples were taken out from the cultures at the indicated time points after α-factor arrest, separated in SDS-7.5% polyacrylamide gels containing 60 μM PhosTag^TM and 120 μM ZnCl₂ and analyzed by Western blotting. Densitometry graphs of lanes 3, 4, 7, 8, 11, 12 were acquired with ImageJ and are shown underneath the lanes. P and arrows indicate the phosphorylated Cac1p-MYC. One of 2 independent experiments with reproducible outcomes is shown.