Skip to main content
. 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 3. Bnip3 induces ER membrane permeabilization.

Figure 3

(A) Left: Immunoblot analysis of GRP94, VAPB, and GAPDH in cytosolic and membrane fractions of Ire1α+/+ and Ire1α knockout (Ire1α−/−) MEFs transduced with adenovirus expressing LacZ or Bnip3 and then treated with TG or untreated (Untx). Right: Quantitation of cytosolic GRP94. (B) Immunoblot analysis of GRP94, VAPB, and GAPDH in cytosolic and membrane fractions of Ire1α−/− MEFs transduced with adenovirus expressing LacZ or Bnip3 at indicated multiplicity of infection (MOI). (C) Co-immunoprecipitation analysis of endogenous Bnip3 and Bcl-2 in Ire1α+/+ and Ire1α−/− MEFs treated with TM or untreated (Untx). Arrows indicate monomer, black arrowheads indicate dimer, and white arrowheads indicate oligomer. (D) Examples of mitochondria and ER fractions of Ire1α−/− MEFs treated with TG or untreated (Untx). Tomm 20 and PDI were used as mitochondrial and ER markers. (E) Gel filtration analysis of endogenous Bak in the ER fractions of Ire1α−/− MEFs transduced with adenovirus expressing LacZ or Bnip3 treated with or without TG. (F) Immunoprecipitation of activated Bak using anti-Bak-N-terminus antibody from the ER fraction of Ire1α−/− MEFs transduced with adenovirus expressing LacZ or Bnip3 treated with TG or untreated (Untx). (G) Immunoblot analysis of GRP78, calreticulin (CRT), VAPB and GAPDH in cytosolic and membrane fractions of wild-type (WT), DKO MEFs (DKO) and DKO MEFs rescued with Bak (DKO + Bak) transduced with adenovirus expressing LacZ or Bnip3 and then treated with TM. (H) Left: Immunoblot analysis of GRP94, Calreticulun (CRT) and GAPDH in cytosolic fractions of Ire1α−/− MEFs stably expressing shRNA directed against Luciferase (shLuc) or Bnip3 treated with TG or untreated (Untx). Right: Quantitation of cytosolic GRP94 and CRT. N=at least 3 biological replicates for (A) to (H). Representative blots are shown. N=3 biological replicates. Statistical significance was calculated by one-way ANOVA followed by Tukey’s test. *: p<0.05. Error bars show S.D.