Removal of Tm from the medium makes the recovery of spliced HAC1 mRNA faster in Ire1-WT cells but not in Ire1-S840D/S841D/T844D cells. (A) Ire1-WT cells incubated with Tm for the amount of time indicated. (B) Quantitation of HAC1 mRNA splicing (closed square). (C) After a 2-h incubation with Tm, Ire1-WT cells were recovered from ER stress by washing them in warm fresh media (Wash) and incubated further without Tm for the remaining time. The white line between lanes 2 and 3 indicates the removal of intervening lanes. (D) Quantitation of HAC1 mRNA splicing for panel C is shown. Blue numbers represent the amount of time after washing. (E–J) Cells were recovered after 1.5 (E and F)-, 1 (G and H)-, and 0.6 (I and J)-h Tm incubation, and the splicing of HAC1 mRNA was quantified. In I, intervening lanes were spliced out between lanes 2 and 3, lanes 3 and 4, and lanes 4 and 5. (K) Ire1-S840D/S841D/T844D cells continue to retain spliced HAC1 mRNA once activated without recovery. (L) The percentage of spliced HAC1 mRNA (closed circle). (M–P) Ire1-S840D/S841D/T844D cells were recovered from Tm after 2 (M and N)- or 6-h (O and P) incubation, and the percentage of HAC1 mRNA splicing was quantified. In M, the splicing of two different parts of a northern membrane is indicated by the white line between lanes 1 and 2. Error bars indicate standard deviations from at least three independent experiments.