FIGURE 6:

Activation steps of Ire1 by DTT imposition and inositol depletion. (A) The ire1Δ strain KMY1516 transformed with an HA-tagged Ire1 plasmid pRS423-IRE1-HA (or empty vector pRS423; no ire1) was cultured in the indicated conditions, and the lysate and anti-HA IP samples were analyzed by anti-HA or anti-BiP Western blotting. (B) Cells used in A (or carrying the W426A mutant version of pRS423-IRE1-HA) were stressed by the indicated conditions and stained by mouse anti-HA antibody/FITC-labeled secondary antibody. (C) The ire1Δ strains transformed with the indicated mutant versions of pRS313-IRE1 were stressed by the indicated conditions, and the total RNA samples were then analyzed by RT-PCR to evaluate splicing efficiencies of the HAC1 mRNA. Error bars represent the SDs from three independent transformants. (D) Cell lysates from the ire1Δ strain KMY1015 transformed with pRS315-IRE1-HA or its mutant versions were analyzed by anti-HA Western blotting.