Figure 2
CRT expression in the WT and Atf6b−/− mice. (A) Expression of Calr mRNA in WT and Atf6b−/− tissues. Total RNA was isolated from the indicated tissues of each mouse and qRT-PCR was performed. n = 5 mice. Data are shown as mean ± SEM. **p < 0.01 by the Mann–Whitney U test. HPC hippocampus, Cx cerebral cortex, SC spinal cord. (B) Expression of CRT protein in WT and Atf6b−/− tissues. Protein samples were extracted from the indicated tissues of WT and Atf6b−/− mice, and subjected to western blotting. n = 5–7 mice. Data are shown as mean ± SEM. **p < 0.01 by the Mann–Whitney U test. (C) Schematic Representation of the promoters used. Triangles indicate the locations and orientations of ERSE motifs that completely or considerably match the consensus CCAATN9CCACG18. Numbers indicate nucleotide positions from transcription start site. ERSE2 and ERSE3 of the human CRT promoter were disrupted by mutating their sequences (marked by crosses). (D) Reporter assays using cultured hippocampal neurons. The CAT ELISA and luciferase assay were performed using cells transfected with the mouse CRT promoters, pCC1, pCC3, and pCC5 (upper graph), or with the human CRT promoters, huCRT(wt) and huCRT(mt) (lower graph). n = 4. Data are shown as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, by a two-way ANOVA followed by the Bonferroni test. (E) Schematic Representation of the full-length (FL), the active form (392) and the dominant-negative form ((392)∆AD) of ATF6β14. AD activation domain, bZip the basic leucine zipper domain, TM transmembrane domain. (F) Reporter assays using cultured WT and Atf6b−/− hippocampal neurons. The CAT ELISA assay was performed using cells transfected with the mouse CRT promoter pCC1. n = 3–5 experiments. Data are shown as mean ± SEM. **p < 0.01, ***p < 0.001, by a two-way ANOVA followed by the Bonferroni test.
