Fig. 4. Stability of Clb2 derivatives in G1-arrested cells. CLB2-HA3 (W1883), CLB2-Δdb-HA3 (W1884) and CLB2-Δ125-HA3 (W1885) were expressed from the GAL10 promoter in cycling or G1-arrested (G1) cdc28-4 bar1-Δ1 cells. Cultures grown at 25°C in raffinose medium were split. One part was further incubated at 25°C (cycling) and induced with galactose for 3 h. The other part was treated with α-factor for 3.5 h and, following arrest in G1, cultures were shifted to 37°C to inactivate Cdk1 (Cdc28) kinase and then induced with galactose. Samples were taken at the indicated times and the protein levels were determined by immunoblotting with HA-specific antibodies (α-HA). Blots were reprobed with an antiserum to β-tubulin (α-Tub2) as a loading control.