Figure 4.—

Clb2-ME diminishes Cdc28-Y19 phosphorylation in vivo. Strains expressing CLB2-HA3 (FHy259) or CLB2-ME-HA3(YGy78) from the endogenous CLB2 promoter were synchronized in G₁ with α-factor and released at 23°. Soluble protein extracts were prepared at the indicated intervals and immunoprecipitated with anti-Ha antibody. (A) Precipitates were analyzed by immunoblotting with anti-PSTAIRE antibody to detect Cdc28 (Cdc28), and anti-phosphotyrosine-Cdc2 antibody to detect specifically Y19-phosphorylated Cdc28 (Cdc28-Y19-P). CLB2 and CLB2-ME samples were run on the same blots for accurate quantification. (B) Quantification of the chemiluminescent signal of Cdc28-Y19-P relative to Cdc28. The values are dimensionless and a value of 1 was assigned for the maximum proportion achieved in the CLB2-ME mutant cells.