Figure 3. Zhangfei suppresses the ability of Xbp1s to activate transcription and requires its leucine zipper to do so.

A and B. Vero cells were transfected with a plasmid containing the coding sequence for CAT linked to a promoter with three copies of the unfolded protein response element as well as a plasmid expressing Xbp1s and varying amounts of plasmids expressing either Zhangfei (ZF) or a mutant, ZF(L/A) in which all leucine residues in the LZip domain had been replaced with alanines. All samples also contained, as a control, a plasmid expressing β-galactosidase. The CAT activity in each sample was normalized to this internal control and expressed as a percentage of the activity in samples containing no vector expressing either ZF or ZF (L/A). The total amount of DNA in each transfection was made up to 5μg with “empty” expression vector (pcDNA3). Bars indicate standard deviation from the mean. B. ZF(L/A) does not activate a promoter containing UPRE but enhances the activity of Xbp1s. C. ZF interacts with Xbp1 with its leucine zipper. Cells were transfected with a vector with the coding sequence for CAT linked to three copies of a sequence, UAS, that binds the DNA-binding domain of the yeast protein GAL4. Cells also received plasmids expressing either ZF or ZF(L/A) linked to the Gal4 DNA-binding domain and either an “empty” expression vector or vectors expressing Xbp1s. Bars represent the ratio of the relative CAT activity (normalized to the internal control, β-galactosidase) of samples with Xbp1s to samples with no activator (“empty” vector). D. An immunoblot showing that vectors with cloned ZF or ZF (L/A) express the proteins in a dose-dependent manner. The results represent the averages of three experiments assayed in duplicate. Bars in all figures represent standard deviation from the mean and p values are indicated on the figures.