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. 2017 Nov 20;46(3):1139–1156. doi: 10.1093/nar/gkx1160

Figure 4.

Figure 4.

Cellular levels of UPR activity and CBC1 mRNA/protein levels bear a reciprocal relationship. (A) Relative Levels of CBC1 mRNA and precursor HAC1 mRNA/HAC1-3′-BE bears a reciprocal relationship. Levels of CBC1 mRNA expressed under the control of a pGAL1-GAL10 promoter, and precursor-HAC1 mRNA were determined by qPCR using RNA harvested from the wild-type yeast strain harboring the engineered pGAL1-GAL10::CBC1 allele at different times after induction with 2% galactose and are presented as means ± SE (n = 3, biological replicates). ER stress was previously imposed with 1 μg/ml tunicamycin, which was added to the culture 3 h before the addition of 2% galactose. The bottom panel of each graph shows the representative gels revealing the relative levels of respective transcripts at different times post-induction as determined by semi-quantitative PCR analysis using cDNA samples prepared from these cells. The arrow denotes the point of induction with 2% galactose. (B) Relative Levels of Cbc1p protein and Hac1p protein bears an inverse relationship. Levels of Cbc1p and Hac1p proteins expressed in wild-type yeast cells expressing a CBC1 allele under the control of pGAL1-GAL10 promoter as determined by western blot analyses using total cellular proteins harvested from a normal strain harboring the engineered CBC1 allele at different times after the induction with 2% galactose. Cells were harvested at different times post-induction as indicated above the panel and total cellular protein extracts were prepared as described and resolved at 12% SDS-PAGE followed by the immunodetection using anti-Cbc1p and anti-Hac1p antibodies. As before, the α-tubulin served as the loading control. (C) Relative levels of transcripts encoding UPR pathway and nuclear exosome/DRN machinery in response to stress induced by DTT (58). The data was retrieved from whole genome expression profile database (www.yeastgenome.org). (D) Relative steady state levels of Cbc1p and Rrp6p proteins in wild type (yBD-117) strains in the absence (–) and in the presence of 5mM DTT (+). α-Tubulin was used as the loading control. Cells were grown in the absence and in the presence of DTT, harvested and total protein extract were prepared as described in Materials and Methods. 50 μg of total cellular protein was loaded in each lane, resolved in 12% SDS-PAGE and probed with anti-Cbc1p, anti-Rrp6p, and anti-tubulin antibodies as described in Materials and Methods.