Figure 3. Amino acid substitutions at the penultimate proline residue affect the half-life of wtTS.

RJK88.13 cells were stably transfected with plasmids encoding wtTS-P2V, wtTS-P2G, wtTS-P2A, wtTS-P2W, wtTS-P2R, wtTS-P2D or wtTS-P2Y. Cells were split into 60 mm plates, and cycloheximide treatment (50 μg/ml) was initiated 24 h later. Cells were harvested after the indicated times of cycloheximide exposure, and steady-state levels of wtTS were determined by Westernblot analysis of total cell extracts using a monoclonal antibody against human TS. As an internal control for equal loading, blots were stripped and steady-state levels of actin were analysed using an α-actin monoclonal antibody; a representative blot for actin is shown (Actin). Half-lives (h), estimated by scanning and quantification of the films, are indicated in parentheses.