FIGURE 2:
UPR inducibility and cellular localization of CPY-GFP and CPY*-GFP. (A) An ire1Δ strain KMY1015 carrying both the wild-type IRE1 (WT Ire1) plasmid pRS315-IRE1-HA and the UPRE-lacZ reporter plasmid pCZY1 was further transformed with the CPY-GFP or the CPY*-GFP expression plasmid (pRS313-TEF1pr-CPY-GFP or pRS313-TEF1pr-CPY*-GFP) or empty vector pRS313 (Vector). The transformant strains were then assayed for cellular β-galactosidase activity, the values of which are normalized against that of vector control cells (set at 1.00). In the “no ire1” sample, cells carried vector plasmids pRS315 and pRS313. Error bars represent the SDs from three independent transformants. According to Student's t test, all values are statistically different from each other (p < 0.05). (B) Total RNA samples from the duplicate transformants used in (A) were subjected to RT-PCR to amplify the HAC1 products, HAC1u (u) and HAC1i (i), which were then fractionated by 13% acrylamide-gel electrophoresis and visualized by fluorescence signal from a fluorescently labeled PCR primer. (C) The transformant strains used in (A) were double-stained with chicken anti-GFP antibody/fluorescein isothiocyanate (FITC)-labeled secondary antibody and rabbit anti–yeast BiP antibody/Cy3-labeled secondary antibody.