Fig 2: Mutant SOD1 proteins accumulate at the MAM and mitochondria in mice and cells expressing mutant SOD1 A, BN2a cells expressing SOD1WT, SOD1G85R or SOD1G93A (A), or spinal cords of WT or mutant SOD1 transgenic mice at end stage (B) were fractionated as described in the Materials and Methods section (Cyto, cytoplasm; P1, nuclei and debris; Mito, mitochondria; MAM, mitochondria‐associated membrane; P3, microsomal fraction). Proper fractionation was confirmed by the fraction‐specific markers as indicated and also shown in Fig EV1A–E. The asterisk denotes non‐specific bands.CTissue‐ and cell type‐specific accumulation of mutant SOD1 at the MAM. Brain, liver, or primary astrocytes from SOD1G93A mice were fractionated and blotted as in (A and B) (upper panel). Quantification of SOD1 proteins at the MAM relative to ones in the cytoplasm was performed from immunoblotting data with an anti‐human SOD1 antibody in (B) and the top panel of (C), and the data are plotted as mean ± SEM from three independent experiments (lower panel). **P < 0.01 versus SOD1WT spinal cord; one‐way ANOVA with subsequent post hoc Tukey's test. Proper fractionation of these samples was confirmed in Fig EV1F–H.DMutant SOD1 prevented association of ER with mitochondria. Isolated ER (P3: microsomal fraction) and mitochondria of N2a cells expressing wild‐type (WT) or G85R (85) SOD1 for 48 h were mixed and incubated. Mitochondrial pellets after centrifugation were analyzed by immunoblotting using anti‐PDI (ER marker) and anti‐VDAC (mitochondrial marker), respectively. All these results were confirmed by three independent experiments. Source data are available online for this figure.

Fig 3: Functional interaction between p38a and ß-catenin in in vitro models of CRC.A In vitro binding assay between GST-p38a fusion protein and HIS-ß-catenin. Bound proteins were analyzed by immunoblotting using anti-GST and anti-HIS antibodies. B Co-immunoprecipitation with anti-HA and anti-FLAG antibodies in HEK293 cells overexpressing HA-p38a or FLAG-ß-catenin. C Co-immunoprecipitation of endogenous p38a and ß-catenin in the indicated cells. D and E Co-immunoprecipitation of endogenous p38a and ß-catenin in nuclear and cytoplasmic fractions of the indicated cells. F Co-immunoprecipitation of endogenous p38a and ß-catenin in nuclear and cytoplasmic fractions from C57BL/6 mice normal colon tissue and AOM-treated APCMin/+ mice adenocarcinoma tissue. Input corresponds to 10% of the lysate. Anti-IgGs were used as negative controls. Lamin B1: nuclear loading control; PDI: cytoplasmic loading control; N = nucleus, C = cytoplasm, ß-cat = ß-catenin.

Fig 4: ER stress in Schwann cells is induced by MPZ mutant overexpression. (A) Western blot analysis demonstrating changes in the levels of ER stress markers (BiP and CHOP) following transfection with wild-type, V169fs and R98C mutants of MPZ in RT4 cells. Quantitative analyses of the western blot analysis data were performed for BiP and CHOP normalized to ß-actin (n=3; *P<0.05). (B) Immunocytochemistry for overexpressed wild-type MPZ and the mutants (Myc), and an ER marker (PDI) indicating the localization of MPZ proteins in relation to the ER in RT4 cells (n=3; scale bar, 10 μm). (C) DCFDA assay showing that expression of both mutants resulted in an augmentation of ROS levels in RT4 cells. Expression of the MPZ V169fs and R98C mutants resulted in statistically significant 2- and 1.2-fold increases in ROS levels, respectively (n=3; *P<0.05; ***P<0.001). MPZ, myelin protein zero.

Fig 5: Activation of UPR by AUY-922 (2 μM) in HuLECWestern Blot analysis of (A) cATF6 and β-actin (B) pIRE1α and IRE1α (C) pPERK and PERK (D) BiP and β-actin (E) ERO1-Lα and β-actin (F) PDI and β-actin after treatment of HuLEC with VEH (DMSO) or AUY-922 (2 μM). The blots represent three independent experiments. Densitometric analysis was performed to evaluate the signal intensity of the proteins of interest. B-actin was used for the normalization of the protein bands, unless otherwise stated. *P < .05, **P < .01, ***P < .001 vs vehicle (VEH). Means ± SEM.