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. Author manuscript; available in PMC: 2015 Jul 6.
Published in final edited form as: Sci Signal. 2015 Jun 23;8(382):ra62. doi: 10.1126/scisignal.aaa0341

Figure 2. IRE1 signaling suppresses ER membrane permeabilization.

Figure 2

(A) Left: Immunoblot analysis of GRP94, GRP78, calreticulin (CRT), VAPB, and GAPDH in cytosolic and membrane fractions of wild-type (WT), Atf6α knockout (Atf6−/−), Ire1α knockout (Ire1α−/−), and Perk knockout MEFs (Perk−/−) treated with TG or untreated (Untx). Right: Quantification of cytosolic GRP78 in WT, Atf6−/−, Ire1α−/−, and Perk−/− MEFs treated with TG or untreated (Untx). (B) Immunoblot analysis of GRP94 and GRP78 (ER luminal), VAPB and IRE1α (ER membrane) and GAPDH (cytosolic) in cytosolic and membrane fractions of WT, Ire1α−/− and Ire1α−/− rescued with WT-IRE1α (Ire1α−/−res.), treated with TG or untreated (Untx). (C) Left: Immunoblot analysis of GRP94, GRP78 and Calreticulin (CRT) (ER luminal), VAPB and IRE1α (ER membrane) and GAPDH (cytosolic) in cytosolic and membrane fractions of INS1 832/13 cells transiently transfected with scrambled control siRNA (si-Cont) or siRNA against IRE1α (si-IRE1α). Right: Quantification of cytosolic GRP78 and Calreticulin (CRT). (D) Immunoblot analysis of GRP94, GRP78, Calreticulun (CRT), VAPB, GAPDH and IRE1α in the cytosolic or membrane fractions of INS1 832/13 cells transduced with LacZ or wild-type IRE1α, treated with TG or untreated (Untx). (E) Quantitation of cytosolic GRP78 and Calreticulin (CRT). (F) Immunoblot analysis of the pro-apoptotic proteins Bax and Bak, the anti-apoptotic proteins Bcl2 and Bcl-xL, the pro-apoptotic BH3 only proteins PUMA, BimEL, Bad, Bid, and Bnip3, in Ire1α+/+ and Ire1α−/− MEFs treated with TM, TG or untreated (Untx). N=at least 3 biological replicates for (A) to (F). Representative blots are shown. Statistical significance was calculated by one-way ANOVA followed by Tukey’s test. *: p<0.05; **: p<0.01. Error bars show S.D.