Figure 2.

Isogenic CDC34 (BL2) and ^tmCDC34 (RC85) yeast strains have similar growth properties, cell size and cell cycle profiles under typical laboratory conditions. (A). Growth on plates. Overnight cultures were adjusted to a density of 1 × 10⁸ cells/ml, serially diluted, spotted onto YPD and grown at 30°C. (B). Growth in liquid culture. Each strain was inoculated in three biological replicates into pre-warmed 50 ml of YPD at a starting density of 5 × 10⁵ cells/ml and grown with vigorous shaking at 31.5°C. The doubling time for CDC34 (BL2) is 86 +/- 2 min and ^tmCDC34 (RC85) is 92 +/- 4 minutes during the exponential phase of growth (0-11.75 hrs). (C). Light microscopic images of cells. Yeast cultures were grown as in B, with images collected at 1.5 × 10⁷ cells/ml (mid-log phase) and 2 × 10⁸ cells/ml (stationary phase). (D). Cell cycle distributions. Yeast cells were analyzed by flow cytometry for their DNA content using propidium iodide staining (Methods). (E). Quantitative western blot analysis of the steady-state levels of Cdc34 and ^tmCdc34 proteins. Yeast extracts were prepared as described in Methods. The indicated amounts of total proteins were separated by SDS-PAGE and analyzed by α-Cdc34 WB. Control α-Skp1 WB was performed to verify equal loading of analyzed samples. Known amounts of purified Cdc34 and ^tmCdc34 were used to verify that the α-Cdc34 antibodies have similar affinity for each protein construct.