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. 2017 Feb;214(2):401–422. doi: 10.1084/jem.20160791

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© 2017 Tschurtschenthaler et al.

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Figure 6. — Selective autophagy receptors target IRE1α for degradation. (A) Ern1 mRNA expression measured by qRT-PCR in ileal scrapings from the indicated genotypes and normalized to Actb expression. n = 15/11/10/11. The mean ± SEM is shown. One-way ANOVA with Bonferroni’s correction was used. (B) IRE1α immunoblot (IB) of Xbp1-sufficient (MODE-K.iCtrl) and Xbp1-deficient (MODE-K.iXbp1) MODE-K cells co-cultured for 24 h with bafilomycin (Baf), an inhibitor of vacuolar H⁺ ATPase that prevents cargo degregation in autophagolysosomes. Data are representative of three independent experiments. (C) IRE1α immunoblot after addition of protein synthesis inhibitor cycloheximide (CHX) and the autophagy inducer rapamycin (Rap) to MODE-K.iCtrl and MODE-K.iXbp1 cells for 24 h. Data are representative of two independent experiments. (D–F) Coimmunoprecipitation (IP) of IRE1α with selective autophagy receptors OPTN (D), NBR1 (neighbor of BRCA1 gene 1 protein; E), and p62 (F) in MODE-K.iCtrl and MODE-K.iXbp1 cells with converse pull-downs also demonstrated. n = 2. Data are representative of three independent experiments. (G) Coimmunoprecipitation of IRE1α with the selective autophagy receptor NDP52 in the human colorectal adenocarcinoma cell line HT29. n = 2. Data are representative of three independent experiments. (H) Coimmunoprecipitation and immunoblot of IRE1α with the selective autophagy receptor NDP52 in HT29 cells at basal conditions (DMSO) and after pharmacological ER stress induction with tunicamycin (Tm). n = 3. Data are representative of two independent experiments. (I) IRE1α and OPTN immunoblots after siRNA silencing of Optn in MODE-K.iCtrl and MODE-K.iXbp1 cells. n = 2. Data are representative of two independent experiments. Ctrl, control. (J) IRE1α immunoblot upon siRNA silencing of Nbr1 or Sqstm1 (p62) in MODE-K.iXbp1 cells. n = 3. Data are representative of two independent experiments. (K) IRE1α and NDP52 immunoblots upon siRNA silencing of NDP52 in HT29 cells with and without tunicamycin treatment for 4 h. n = 2. Data are representative of two independent experiments. (L) Coimmunoprecipitation of OPTN with IRE1α in ileal crypts of Wt and Xbp1^ΔIEC mice. n = 3. Data are representative of two independent experiments.