Figure 8.

Characterized ER stress-sensing pathways do not regulate ERAD-T in the presence or absence of ER stress. A, C, and D, cycloheximide chase analysis of yeast of the indicated genotypes expressing Deg1*-Sec62 or harboring an empty vector (Vec) cultured in the presence or absence of 6 mm DTT for 1 h. DTT was maintained at the same concentration during incubation with cycloheximide. Deg1*-Sec62 was detected with peroxidase anti-peroxidase antibodies. Pgk1 served as a loading control. The percentage of Deg1*-Sec62 remaining at each time point (normalized to Pgk1) is indicated below the images. IRE1 (A), SLT2 (A), EMP24 (C), and UBR1 (D) gene deletions were verified by PCR genotyping.⁸ B, yeast of the indicated genotypes were incubated in the presence or absence of 6 mm DTT for 1 h prior to RNA extraction and RT-PCR to analyze HAC1 mRNA splicing. Expected product sizes are 969 bp for unspliced HAC1 (HAC1(u)) and 717 bp for spliced HAC1 (HAC1(s)). Experiments depicted in A, C, and D were performed three times (with the exception of the right panel of A, which was performed two times). The experiment depicted in B was performed one time.