Anti calnexin

Western blot analysis was carried out using standard methods. Briefly, cells and EV pellets were lysed on ice with lysis buffer (150 mmol/L NaCl, 50 mmol/L TRIS pH 7.4, 0.25% NP40, 1mM EDTA, 0.1% Triton X100, 0.1% SDS, 0.1 mg/mL phenylmethylsulfonyl fluoride, protease cocktail inhibitor (Merck Life Science, Milan, Italy; cat. no.04693159001), 50 mmol/L NaF, and 10 mmol/L Na₃O₄V. For Western blots, equal protein concentrations (quantified with the Bicinchoninic Acid (BCA) method, Merck Life Science, Milan, Italy; cat. no. B9643) were resolved via 12% SDS–PAGE and transferred to PVDF membranes (Biorad Laboratories S.r.L. Rome, Italy). Antibodies used were: anti-ALIX (Covalab, Bron, France; cat. no. PAB 0204, 1:1000); anti-TSG101 (Gene Tex, Irvine, CA, USA; cat. no. GTX70255); anti-calnexin (Enzo, Farmingdale, NY, USA; cat. no. ADI-SPA-860-F 1:1000); anti-GFP (Merck Millipore, Milan, Italy; cat. no. 3580, 1:1000); goat anti-rabbit IgG, HRP (Abcam, Cambridge, UK; cat. no. 6721) or goat anti-mouse IgG, HRP (Abcam, Cambridge, UK; cat. no. 6728), 1:5000. ECL Western blotting substrate (GeneTex, Irvine, CA, USA; Trident fento Western HRP substrate, cat. no. GTX14698) was added before detection with a BioRad Chemidoc XRF+ (Biorad Laboratories S.r.L. Rome, Italy). Full blots are shown in Figure S6.

Mice were deeply anaesthetized and transcardially perfused with phosphate-buffered saline (PBS) followed by 4% paraformaldehyde (PFA) in 0.1 M phosphate buffer (PB). Then, the brains were removed and postfixed in the same fixative for 2 h at 4 °C and subsequently cryoprotected in 30% sucrose in PB. Frozen sections, either 10 or 50 μm thick, were prepared on a microtome. Sections were washed with PBS and incubated for 1 h at room temperature in blocking buffer: 20% Block Ace (KAC Co., Ltd.), 5% normal goat serum (NGS), 0.1% Triton X-100, 0.1% azide in PBS. Then, the sections were incubated overnight with primary antibody in antibody dilution buffer (5% Block Ace, 5% NGS, 0.1% Triton X-100, 0.1% azide in PBS) at 4 °C. Sections were then washed with 0.1% Triton X-100 in PBS and incubated for 1 h with secondary antibody in antibody dilution buffer at room temperature. The antibodies used were as follows: anti-calbindin (1:500; Sigma-Aldrich), anti-CTCF (1:1000; Cell Signaling Technology), anti-VGluT2 (1:10,000; Millipore), anti-active caspase-3 (1:500; Cell Signaling Technology), anti-calnexin (1:500; Enzo), anti-KDEL (1:2000; MBL), and anti-IP3R (1:500; abcam). For haematoxylin and eosin (HE) staining, sections were stained with Mayer’s haematoxylin and eosin Y (Muto Pure Chemicals, Tokyo, Japan).

Human embryonic kidney 293 T (HEK293T) cells were cultured as previously described and transfected using polyethylenimine. For western blotting analysis, cells were washed with ice-cold PBS and scraped in 100 ml lysis buffer (150 mm NaCl, 2% sodium dodecyl sulfate, 10 mm Hepes pH 7.4, 2 mm EDTA) plus protease inhibitor cocktail (Roche Diagnostics). Total lysates were sonicated and centrifuged at 13,000 rpm for 10 min at 4 C. Cells lysates were denatured at 95 C in sample buffer and processed for 4–12% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE), and electro-transferred onto nitrocellulose membranes (Millipore). Immunoblotting was done in 5% non-fat dry milk dissolved in Tris-buffered saline using the following antibodies: anti-FUS (1:2000, sc-25-540, Santa Cruz); anti-Tubulin (1:10000, Sigma); anti-Calnexin (1:5000, Enzo); anti-Laminin B1 (1:1000, Abcam). Immunoreactivity was detected using IRDye-conjugated Goat Anti-Rabbit or Anti- Mouse IgG (Li-Cor), and visualized using Odissey Imaging System (Li-Cor). Experiments were ran in triplicate using three independent lysate preparations from cultured cells.

Immunoblotting analysis was carried out according to the standard procedure (Sambrook et al., 1989 ) as described previously (Ninagawa et al., 2011 (link)). Chemiluminescence obtained using Western Blotting Luminol Reagent (Santa Cruz Biotechnology) was detected using an LAS-3000mini LuminoImage analyzer (Fuji Film). Rabbit monoclonal anti-TXNDC11 antibody was obtained from Abcam. Rabbit polyclonal anti-EDEM2 antibody was obtained from Novusbio; and anti-HA from Recenttec. Rabbit polyclonal anti-calnexin, anti-PDI, and anti-ERp72, and anti-calreticulin antibodies were obtained from Enzo Life Sciences. Mouse monoclonal anti-Flag antibody was obtained from Sigma; and anti-β-actin from Wako. Anti-human ATF6α antibody was produced previously (Haze et al., 1999 (link)).
Immunoprecipitation was performed using anti-Flag antibody and protein G-coupled Sepharose beads (GE Healthcare). Beads were washed with high salt buffer (50 mM Tris/Cl, pH 8.0, containing 1% NP-40 and 150 mM NaCl) twice, washed with PBS, and boiled in Laemmli's sample buffer.

Reagents were as follows: ethylene glycol tetraacetic acid (EGTA) (Acros Organics, Geel, Belgium, 409910250), Fura-2 AM (Biotium, Kampenhout, Belgium, 50033), Annexin V-Fluorescein isothiocyanate (FITC) (Becton Dickinson, Franklin Lakes, NJ, USA, 556419), 7-aminoactinomycin D (7-AAD) (Becton Dickinson, 555815), U73122 (Enzo Life Sciences, Farmingdale, NY, USA, BML-ST391-0005), U73343 (Enzo Life Sciences, BML-ST392-0005), venetoclax (ChemieTek, Indianapolis, IN, USA, CT-A199), anti-human IgG/M (Jackson ImmunoResearch, West Grove, PA, USA, 109-006-127). The following antibodies were used: anti-IP₃R2 (Abiocode, Agoura Hills, CA, USA, R2872-3); anti-calnexin (Enzo Life Sciences, Farmingdale, NY, USA, ADI-SPA-865-D); anti-Bcl-2 (Santa Cruz Biotechnology, Dallas, TX, USA, sc7382HRP); anti-Bim (Bioké, Leiden, The Netherlands, 2819 S); anti-GAPDH (Sigma-Aldrich, St. Louis, MO, USA, G8795); anti-vinculin (Sigma-Aldrich, Munich, Germany, V9131). The sequences of the peptides used in this study were: BIRD-2 (RKKRRQRRRGGNVYTEIKCNSLLPLAAIVRV) and TAT-Ctrl (RKKRRQRRRGGSIELDDPRPR). These peptides were synthesized by LifeTein (South Plainfield, New Jersey, USA) with a purity of at least 85%. The IP₃ sponge (pEF-GSTm49-IRES-GFP) is a protein constructed from the IP₃-binding core of the type 1 IP₃R with a single amino acid substitution (R441Q) that has a very high affinity for IP₃ [29 (link)].