Figure 6.

Degradation of Deg1-GFP is unaffected by the hrd4–1 mutation. (A) Cycloheximide chase of Deg1-GFP was performed by the addition of cycloheximide to log-phase cells at the indicated times before collection. Equal numbers of cells were lysed and analyzed by SDS-PAGE, followed by immunoblotting for GFP with the use of anti-GFP antibody. (B) Loss of Deg1-GFP immunoreactivity during cycloheximide chase. Densitometric analysis of data obtained in (A) was used to determine loss of GFP immunoreactivity in each indicated strain at 0, 30, and 60 min. (C) Steady-state fluorescence of the Deg1-GFP protein was determined for the indicated strains by flow cytometry. Fluorescence was identical for the following strains: HRD4⁺ Deg1-GFP, hrd4–1 Deg1-GFP, and a HRD4⁺ strain transformed with no GFP construct at all (cell autofluorescence). All experiments were performed at the permissive temperature of 30°C.