Figure 1. SEC63 deficiency activates IRE1α-XBP1 but not other UPR branches.

(A) Xbp1 mRNA RT-PCR splicing assay in Sec63- or Prkcsh-deficient cells and kidney tissues. Spliced XBP1s is only present in SEC63-KO cells and kidneys. (B) WT (Sec63^fl/fl), SEC63-KO cells, and SEC63-KO cells reconstituted with SEC63 or GFP in the unstressed state or were treated with tunicamycin (Tun; 2 μg/ml) or thapsigargin (Thap; 1 μM) for 4 hours. IRE1α and PERK activation (phosphorylation) was analyzed by immunoblotting. Phos-tag electrophoresis was performed to better separate p-IRE1α from the unphosphorylated form. (C) WT and SEC63-KO cells in the basal state or treated with tunicamycin for the indicated times. UPR activation analyzed by immunoblotting with indicated antibodies. (D and E) Quantitative RT-PCR expression of UPR marker genes in SEC63-KO and the reconstituted cells (D, n = 3 per group), and SEC63-KO P18 kidneys (E, n = 6–8 mice per group). (F) Kidney nuclear extracts examined by immunoblotting with indicated antibodies. Tunicamycin-treated (1 mg/kg, 6 hours) mice were used as positive controls. ns, a nonspecific band used for loading control. (G) eIF2α phosphorylation was determined by immunoblot using whole kidney lysates. Note the residual SEC63 expression in SEC63-KO kidneys due to the cell type–specific expression of Ksp-Cre. Results are shown as mean ± SEM (Student’s t test); ***P < 0.001; **P < 0.01, *P < 0.05.