(a) SV40 Large T antigen-immortalized mouse embryonic fibroblasts were transduced with the indicated knockdown constructs and treated with thapsigargin (TG). Ifnb mRNA induction was evaluated using quantitative RT-PCR at the indicated time points. Data are representative of 3 experiments.
(b) Primary mouse macrophages were transduced and treated as in (a).
(c) Endonuclease activity of IRE-1 is responsible for removal of an intron in XBP-1 mRNA, as well as destructive cleavage of ER-localized RNAs. The kinetics of UPR activation were assessed using a splicing assay for XBP-1 mRNA. The illustration shows the PstI digest site in the intron of unspliced XBP-1 mRNA used to distinguish spliced (S) and unspliced (U1, U2) cDNAs.
(d–e) Primary mouse macrophages were treated with thapsigargin (TG; e) or tunicamycin (TM; f) treatment. *P < 0.05; **P < 0.0001 (2-way ANOVA, b–e). Data are representative of at least three independent experiments with biological triplicates (mean + s.d., b–e).