Fig. 4.
Human IRE1α mutant D123P is defective in dimerization, autophosphorylation, and RNase activity. (A) COS-1 cells were cotransfected with plasmids expressing human IRE1α (WT or D123P) tagged with Flag and HA epitopes. Lysates were prepared from cells treated with or without Tm for 4 h and subjected to immunoprecipitation using anti-Flag monoclonal antibody and then analyzed by Western blot using anti-HA antibody. (B) Ire1α−/− MEF cell lines stably expressing human IRE1α WT or mutant proteins were treated with or without Tm for 5 h. Cell lysates were prepared and subjected to immunoprecipitation by using anti-IRE1α antibody and then analyzed by Western blot with antibodies against IRE1α. (C) Quantitative real-time RT-PCR analysis of spliced Xbp1 transcripts. WT and Ire1α−/− MEF cell lines stably expressing WT or mutant human IRE1α proteins were treated with Tm (10 μg/ml) for 5 h, and RNA was isolated for quantitative real-time RT-PCR by using a primer set flanking the intron in the Xbp1 mRNA. The columns and bars represent the means and standard deviations of three independent experiments.