Figure 8.

S724A mutation of IRE1α results in blunted XBP1s production with decreased PDI but increased CHOP expression in ER-stressed livers.A–D, immunoblot analysis of the phosphorylation and expression levels of the indicated proteins in livers of Ern1^S724A/S724A mice and their Ern1^WT/WT littermates following treatment with Tm or vehicle for 6 h (A and B) or 24 h (C and D). Shown are representative immunoblots for three individual mice per group (A and C), and quantification of XBP1s, PDI, and CHOP protein levels after normalization to tubulin (B and D). E and F, primary hepatocytes from male Ern1^S724A/S724A mice and their Ern1^WT/WT littermates were infected for 30 h with adenoviruses expressing GFP control or XBP1s protein. Cells were then treated with Tm (100 ng/ml) for 24 h. E, immunoblot analysis of XBP1s protein, IRE1α phosphorylation, as well as PDI and CHOP proteins. Shown also is the quantification of PDI and CHOP protein levels after normalization to tubulin as the loading control. F, quantitative RT–PCR analysis of the mRNA abundance of the indicated genes. Results in (E) and (F) represent three independent experiments. All data are presented as the mean ± SD. ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001 by two-tailed unpaired Student’s t test or two-way ANOVA. CHOP, CCAAT-enhancer binding protein homologous protein; ER, endoplasmic reticulum; IRE1α, inositol-requiring enzyme 1α; PDI, protein disulfide isomerase; Tm, tunicamycin; XBP1s, spliced X-box binding protein 1.