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. Author manuscript; available in PMC: 2015 Jul 31.
Published in final edited form as: Cell. 2014 Jul 10;158(3):534–548. doi: 10.1016/j.cell.2014.07.002

Figure 4. KIRA6 inhibits IRE1α Terminal UPR outputs and apoptosis.

Figure 4

(A) Anti-total JNK, anti-phospho-JNK, and anti-Pro- and Cleaved Caspase-3 immunoblots of INS-1 IRE1α (WT) cells treated with Dox (5 ng/ml) −/+ 1μM KIRA6 for 72hr. (B) JNK2α1 phosphorylation under indicated [KIRA6] by in vitro ELISA-based anti-phospho-JNK assay. (C) Anti-total and phospho-JNK immunoblots of INS-1 cells pretreated for 1hr with indicated [KIRA6], then 1μM Tg for 2hr. (D) Q-PCR for Ins1 mRNA in INS-1 IRE1α (WT) cells treated with Dox (5 ng/ml) −/+ KIRA6 (1 μM). (E) Anti-Proinsulin immunoblot of samples in (A). (F) % Annexin V staining in INS-1 IRE1α (WT) cells after 72hr in Dox (5 ng/ml) and indicated [KIRA6]. (G) Competition between indicated [1NM-PP1] and KIRA6 (1 μM) for IRE1α* (I642G) RNase. (H) Agarose gel of PstI-digested XBP1 cDNA amplicons from INS-1 cells IRE1α (I642G) cells induced by Dox (1 μg/ml) for 24hr, then 1NM-PP1 (0.5 μM) −/+ indicated [KIRA6] for 3hr, with quantitation. (I) Annexin V staining of INS-1 IRE1α (I642G) cells after 72hr with Dox (1 μg/ml), Tm (0.5 μg/ml), 1NM-PP1 (1 μM) and indicated [KIRA6]. (J) Model of 1NM-PP1 and KIRA6 competition of oligomerization and RNase activity in IRE1α* (I642G). Data plotted as mean +/− SD. P-values: *<0.05, ** <0.01. Also see Figure S4.