Figure 3

Expression of molecular chaperones in the ER and Ca²⁺ levels in WT and Atf6b^−/− hippocampal neurons. (A) Total RNA was isolated from WT and Atf6b^−/− hippocampal neurons (n = 3–8), and qRT-PCR was performed with the indicated primers. Data are shown as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001 between two genotypes by a two-way ANOVA followed by the Bonferroni test. Note that expression of Calr mRNA was significantly lower in Atf6b^−/− neurons than in WT neurons under both normal and ER stress conditions. (B) Protein samples extracted from WT and Atf6b^−/− hippocampal neurons exposed to control or ER stress conditions for 16 h (n = 5–6) were analyzed by western blotting using antibodies against the indicated proteins. Data are shown as mean ± SEM. ***p < 0.001 between two genotypes and ^#p < 0.05, ^##p < 0.01, ^###p < 0.001 compared to normal conditions by a two-way ANOVA followed by the Bonferroni test. Note that expression of CRT protein was significantly lower in Atf6b^−/− neurons than in WT neurons under both normal and ER stress conditions. (C) Ca²⁺ levels in the ER, cytosol and mitochondria of hippocampal neurons were measured using G-CEPIA1er, GCaMP6f. and CEPIA2mt, respectively under normal and ER stress (Tm for 3 h) conditions. n = 60–250 cells in each condition from two independent experiments. Data are shown as mean ± SEM. ***p < 0.001 between two genotypes and ^#p < 0.05, ^##p < 0.01, ^###p < 0.001 compared to normal conditions by a two-way ANOVA followed by the Bonferroni test.