Def1 is required for translation. (A) DEF1 null cells are sensitive to translation inhibitor, hygromycin B. Cells were spotted onto YPAD media, and the same was done with YPAD containing 50 µg/mL hygromycin. Images were captured at 24 and 48 h. (B) Puromycin incorporation is impaired in def1Δ. Cells were grown to log phase, and puromycin was added for 15 min before harvesting. Puromycin incorporation was detected by western blot analysis of cell extracts using antipuromycin antibody. Results are the average and standard deviation of biological triplicates (N = 3). (C) Representative polysome profiles of wild-type and def1Δ cells overlaid (wild-type cells in black and def1Δ in red). The average polysome-to-monosome ratios (P/M) were determined and represented in the table. The area under the curve (AUC) for the monosome (80S) and polysome fractions was calculated using PeakChart 3 software (Brandel). The average and standard deviations (N = 3) of independent experiments are shown.
