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 tm
CDC34 (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.