The nuclease activity of Ire1 is required for RIDD. (A) Western blot of Flag-tagged hIre1 proteins. GAPDH, glyceraldehyde 3-phosphate dehydrogenase. (B) Ethidium bromide–stained agarose gel showing XBP-1 reverse transcribed from total RNA and PCR amplified using primers surrounding the splice site. The asterisk denotes a hybrid band amplified from spliced and unspliced (u) products (Shang and Lehrman, 2004). s, spliced product after the removal of the 26 nucleotide intron. Bands were quantified in ImageJ (National Institutes of Health) to determine the percentage of splicing. (C and D) RNA levels of RIDD targets and controls in DTT (C)- and Tm (D)-treated cells measured by qPCR and normalized to signals from Rpl19. (B–D) The means and SDs for three independent experiments are shown.