Fig. 4.

Feedback activation of IRE1α by XBP1 deficiency, and IRE1α-mediated mRNA cleavage. (A) Western blot and (B) Quantitative RT-PCR analysis of Min6 cells expressing luciferase or XBP1 shRNAs. (C) 293T cells were cotransfected with EGFP, Ins1, PC1, PC2, and CPE plasmids together with WT or K599A mutant IRE1α. Cells were harvested 24 h after transfection for quantitative RT-PCR analysis. EGFP mRNA levels were used for normalization. Values represent fold changes relative to vector controls. (D) Total Min6 RNA was incubated with increasing amounts of recombinant IRE1α and then separated on an agarose gel. Northern blot was performed using the indicated ³²P-labeled probes. The asterisks indicate cleavage products.