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. 2019 Jan 8;10(3):263–276. doi: 10.18632/oncotarget.26467

Figure 3. Smc4 protein is degraded in mitosis.

Figure 3

A. Upper panel: Western blot analysis of Smc4 protein level in SMC4-HA and GAL-SMC4-HA strains. Overnight cultures were grown in medium containing galactose (YPG medium). After diluting, the SMC4-HA culture was maintained in medium with galactose for 3 hours, whereas the GAL-SMC4-HA culture was grown in medium containing galactose (YPG) or dextrose (YPD). PSTAIRE recognizes Cdc28 protein as the loading control. Middle panel: Replica patching assay indicating GAL-SMC4-HA viability on rich medium with galactose (left) and lethality on dextrose (right) at RT, 30°C, 37°C. Bottom panel: Colony survival of wild type, SMC4-HA and GAL-SMC4-HA strains plated on galactose containing rich medium. Cells in log phase (1 OD) in rich liquid (YPG) medium were counted and 500 cells were plated in six replicates. Number of surviving colonies was determined after 5 days at 30°C. B. Analysis of a synchronized cell cycle after G1 arrest of the GAL-SMC4-HA strain and release into galactose or dextrose containing rich medium. After release, samples were taken every 10 minutes and subjected to Western blot analysis for Smc4 protein level. PSTAIRE recognizes Cdc28 protein as the loading control. The cell cycle stage labels show the approximate cell cycle timing based on the known relationship between budding, DNA replication and mitosis. C. Analysis of a synchronous cell cycle after G1 arrest in rich (YPG) medium in wild type and GAL-SMC4-HA strains. After releasing from G1 arrest, samples were taken for scoring budding (green), chromosome condensation (red) and anaphase (black) as described in Figure 1.