Fig. 3. Pkc1p is required for the maintenance of Cln2p. (A) Oscillation of Swe1 or Cln2 protein levels during cell-cycle progression in various strains that were synchronized with -factor was determined by western blot analysis. Cells in early log-phase (OD₆₀₀ of 0.20.3) of wild-type (YMM180), pkc1-834 (YMM179), zds1 (YMM187), zds1 pkc1-834 (YMM178) and stt1-1 (YMM196) strains, with a chromosomally integrated construct encoding genomic copies of Swe1p-9xMyc and Cln2p-3xHA, were synchronized with -factor in G₁, and resuspended in YPD or YPD plus 100 mM CaCl₂ at 25°C. Samples were taken at of 20-minute intervals after release from arrest. Cdc28 protein was used as an internal loading control. (B) Stability of Cln2p. Wild-type (YMM180), pkc1-834 (YMM179) and stt1-1 (YMM196) cells were cultured at 37°C for 2 hours; and then samples were taken at the indicated times after the addition of 100 µg/ml cycloheximide, and subjected to western bolt analysis. The asterisk indicates a non-specific band. (C) DNA content for the same samples as in A. (D) Effect of over-expression of CLN2 on the bud growth of the zds1 pkc1-834 strain. A strain containing a chromosomally integrated construct for GAL-regulated Cln2p-3xHA (YMM170-1; cln2::GAL1–CLN2^HA–LEU2) were grown in the medium containing 2% raffinose (GAL1 promoter off) or 2% galactose (GAL1 promoter on) for 8 hours at 25°C.