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Fig. 9. ER-luminal domain and cytoplasmic domain of xIRE1β show different effects on embryogenesis and xXBP1 splicing and have different subcellular localizations. (A) Diagram depicting the construction of deletion mutants. S and TM denote the signal peptide and the transmembrane domain. (B) An uninjected control embryo at tailbud stage. (C and D) Embryos injected with 1 ng xIRE1βδC (C) and 500 pg xIRE1βδN (D) caused phenotypic changes. (E) Expression of Xbra was repressed by injection of xIRE1βδC (middle) but not by injection of xIRE1βδN (right). (F) Xsox17α was not affected significantly by injection of either xIRE1βδC (middle) or xIRE1βδN (right). (G) Injection of xIRE1βδC resulted in decrease of cytoplasmic variant of xXBP1 in embryos collected at indicated stages while injection of xIRE1βδN caused an increase. (H) Monitoring of subcellular localization and splicing activity of the xIRE1β-specific EGFP fusion constructs xIRE1β (1–958, upper panel), xIRE1βδC (1–549, middle panel) and xIRE1βδN (439–958, lower panel) in HeLa cells. Both, xIRE1β and xIRE1βδC show a similar subcellular localization but xIRE1βδC the did not activate the xXBP1(U)-RFP sensor (middle panel). The xIRE1βδN mutant is active but shows a different subcellular localization (lower panel). |
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Fig. 3. Overexpression of xIRE1β does not affect mesoderm and endoderm formation but enhances cleavage of xXBP1 pre-mRNA. (A) Uninjected control embryo at tailbud stage. (B) Injection of xIRE1β at 1.5 ng. (C and D) Overexpression of xIRE1β did not change the expression of Xbra (C) and Xsox17α (D) at stage 10.5. (E) Overexpression of xIRE1β resulted in increase of cytoplasmic splicing of xXBP1. ODC (ornithine decarboxylase) was used as loading control. xXBP1(U), unspliced xXBP1; xXBP1(N), nuclear spliced xXBP1. |
