Cells from 5- to 10-ml aliquots of meiotic cultures were harvested, and 1 ml of 20% trichloroacetic acid (TCA) was added. The supernatant was removed after centrifugation, and the pellet was resuspended in 100 μl of 20% TCA and stored al −80°C. Samples were thawed on ice, glass beads were added, and cells were broken using a FastPrep FP120 cell disrupter (BIO 101 ThermoSavant, Obiogene, Carlsbad, CA). The lysate was recovered by punching a hole on the bottom of the tube, and the glass beads were further washed with 200 μl of 5% TCA. Lysates were centrifuged at 1000 × g for 3 min, and the pellet was thoroughly resuspended in 100 μl of 2× Laemmli buffer and 50 μl of 2 M Tris base. After boiling for 5 min, 10–20 μl were loaded in the gels. Ndt80 and Cdc5 production was analyzed in 10% SDS–PAGE gels with a 37.5:1 ratio of acrylamide:bisacrylamide. To resolve the phosphorylated forms of Mek1, 10% gels (acrylamide:bisacrylamide 29:1) containing 37.5 μM Phos-tag reagent and 75 μM MnCl2 were used. After running, Phos-tag gels were washed with 1 mM EDTA before transfer. The anti-Ndt80 (kindly provided by K. Benjamin) and anti-Cdc5 (sc-6733; Santa Cruz Biotechnology, Santa Cruz, CA) antibodies were used at 1:5000 and 1:500 dilutions, respectively. To detect phosphorylation of Cdc28 at tyrosine 19, anti–phospho-Cdc2(Tyr15) (Cell Signaling Technology, Beverly, MA) was used at 1:1000 dilution. Total Cdc28 was detected with anti-PSTAIRE (sc-53, 1:500 dilution; Santa Cruz Biotechnology,). The anti-Mek1 antibody was previously described (Refolio et al., 2011 ). Anti-tubulin (TAT1; 1:5000 dilution) and anti–phosphoglycerate kinase (PGK) (A-6457, 1:5000 dilution; Molecular Probes, Invitrogen, Carlsbad, CA) were used as loading controls. The ECL or ECL+ reagents (GE Healthcare, Piscataway, NJ) were used for detection. The signal was captured with a ChemiDoc XRS system (Bio-Rad, Hercules, CA), using the Quantity One software.