Figure 4.

Steady state levels and half-life of Tra1${}_{\text{Q}3}$. (A) Yeast strains CY6808 (Flag⁵-tagged TRA1; lanes 1-3), CY5920 (Flag⁵-tagged TRA1 with HIS3 inserted downstream of the TRA1 locus; lanes 4-6), CY6605 (Flag⁵-tagged tra1${}_{\mathit{Q3}}$ with HIS3 inserted downstream of the TRA1 locus; lanes 7-9) and BY4742 (No Tag, lane 10) were grown to stationary phase in media lacking leucine, diluted 1:20 in YPD and grown for eight hours. Protein lysates were prepared by bead lysis and 40 μg, 20 μg, and 10 μg of total protein was loaded, except for the no tag control where 40 μg was loaded. Extracts were separated by SDS-PAGE and blotted with anti-Flag antibody. The bottom of the gel was stained with Coomassie Brilliant Blue (CBB) as a loading control. (B) Yeast strains expressing Flag⁵-tagged Tra1${}_{\text{WT}}$ (lane 1-5; CY6808) or Tra1${}_{\text{Q}3}$ (lane 6-10; CY6605) were grown to saturation in media lacking leucine, diluted 1:25 and grown for 6 hr. Translation was inhibited with 35 μg/ml cycloheximide. Cells were harvested at 0, 2, 4, 6 and 8 hr after cycloheximide addition and 40 μg of total protein from each time point was separated by SDS PAGE and blotted with anti-Flag antibody. The bottom of the gel was stained with CBB as a loading control. (C) TRA1 (CY4353) and tra1${}_{\mathit{Q3}}$ (CY6582) were grown to saturation in media lacking leucine, diluted 1:10 in YPD and grown for eight hours. Transcript levels of TRA1 were measured and normalized to U3 (snR17a) as a loading control. Data represent mean values $\pm$ SD from three independent biological replicates.