The VP16 activation domain, but not other domains that activate transcription, confers protein instability. (A) Steady-state levels of GAL4-fusion activators. Human HeLa cells were transiently transfected with expression constructs encoding the indicated GAL4–fusion activators (lanes 2–9) or with pUC119 carrier DNA (lane 1). After transfection, cells were treated with either DMSO solvent (−PS1) or PS1 (+PS1). Cells then were harvested, and equal volumes of total cell lysates were resolved by SDS/PAGE. GAL4-fusion proteins were detected by immunoblotting. (B) Stability of GAL4-fusion activators. The indicated GAL4-fusion proteins were transiently expressed in HeLa cells and pulse–chase analysis was performed as described (1). Labeled GAL4–fusion proteins were recovered by denaturing immunoprecipitation, and visualized by SDS/PAGE followed by autoradiography.