Figure 9.

Kar2 overexpression does not rescue impaired ERAD-T during ER stress. Cycloheximide chase analysis of WT yeast expressing Deg1-Sec62 and harboring low-copy (centromeric) (A) or high-copy (2μ) (B) plasmids encoding Kar2 (or matching empty vector controls) cultured in the presence of 6 mm DTT or no treatment for 1 h. DTT was maintained at the same concentration during incubation with cycloheximide. Deg1-Sec62 was detected with AlexaFluor-680–conjugated rabbit anti-mouse antibodies. Kar2 was detected with anti-Kar2 antibodies. Pgk1 served as a loading control. The percentage of Deg1-Sec62 remaining at each time point (normalized to Pgk1) and Kar2 steady-state abundance relative to Empty Vector/No Treatment controls (normalized to Pgk1) are indicated below the images. Experiments depicted were performed three times.