Regulated Ire1-dependent decay of messenger RNAs in mammalian cells

Hollien, Julie; Lin, Jonathan H.; Li, Han; Stevens, Nicole; Walter, Peter; Weissman, Jonathan S.

Online supplemental material Fig. S1 shows the array data for hIre1 / and hIre1 R cell lines, a comparison with published data for UPR targets, and confirmation of RIDD target regulation measured by qPCR. Fig. S2 shows decay rate measurements for 11 RIDD candidates in the presence and absence of DTT. ferritin-like protein recombination target (FRT) sites for site-specific transgene expression by transfection with pFRTlacZeo (Invitrogen), and stable clonal integrants were selected with Zeocin . We then transfected the Ire1- / FRT cells with the pOG44 ferritin-like protein recombinase vector (Invitrogen) and FRT vectors containing hIre1 under the control of the human ER-1 promoter (Lin et al., 2007) . Quik-Change mutagenesis (Agilent Technologies) was used to make the point mutations in the hIre1 coding sequence. Multiple independent isogenic clones were analyzed for transgene mRNA and protein expression with identical findings. The ATP analogue 1NM-PP1 was a gift from C. Zhang and K. Shokat (University of California, San Francisco, San Francisco, CA; Bishop et al., 2000) .