Anti calreticulin

Protein was extracted as described [52 (link)], quantitated using BCA assay (Pierce) and subjected to immunoblot analysis using anti-Bmi1 (Abcam; Cambridge, MA), anti-CHOP, anti-calreticulin, anti-phospho-EIF2α, anti-β-tubulin (Cell Signaling, Danvers, MA) and anti-Grp78 (Abcam). Secondary antibodies were from Santa Cruz Biotechnology (Dallas, TX). Molecular weight markers (Cat. # 10748010, 5 µL per run, or Cat. #LC5800, 10 µL per run) for immunoblot analyses were from Invitrogen (Grand Island, NY).

Cell lysates and brain homogenates were prepared in PBS supplemented by a cocktail of protease inhibitors. Protein concentration was determined with the Pierce BCA Protein Assay kit (Thermo Scientific). Proteins from cell lysates or brain homogenates were fractionated by electrophoresis using 4–12% SDS-polyacrylamide gels (SDS-PAGE), transferred into nitrocellulose membranes, and probed with the following antibodies: anti-prion 6D11 antibody (1:5000, Sigma), anti-calreticulin (1:1000, Cell Signaling), anti-GRP94 (1:1000, Cell Signaling), anti-protein disulphide isomerase (anti-PDI, 1:1000, Cell Signaling), anti-protein kinase RNA-like endoplasmic reticulum kinase (PERK) (1:1000, Cell Signaling), anti-phospho-PERK (1:1000, Cell Signaling), anti-inositol-requiring 1 protein (IRE1) (1:1000, Cell Signaling), anti-eIF2α (1:1000, Cell Signaling), anti-phospho-eIF2α (1:1000, Cell Signaling), anti-calnexin (1:1000, Cell Signaling), anti-CCAAT/-enhancer-binding protein homologous protein (CHOP) (1:1000, Cell Signaling), and anti-β-actin (1:1000, Cell Signaling). The immunoreactive bands were analyzed using the Quantify One (4.6.7) software (BioRad^®).

MSCs were lysed and the protein concentration was determined using the Pierce BCA Protein Assay Kit. Aliquots (40 μg) of protein solutions were resolved by 10% SDS-PAGE (Millipore, Billerica, MA) and transferred to PVDF membranes. The membranes were incubated with diluted anti-RUNX2, anti-PPARγ, anti-GAPDH, anti-β-catenin (ProteinTech), anti-CD9, anti-CD81, anti-Fas, anti-integrin alpha-5, anti-CD44, anti-p65 (Abcam), anti-calreticulin (Cell Signaling Technology), anti-AKT1 (Santa Cruz), anti-p-AKT1, anti-ERK and anti-p-ERK (Abclonal) primary antibodies, followed by the corresponding secondary antibodies (Cell Signaling Technology). Bands were detected using the ECL Kit (NCM bio, Suzhou, China). Images were analyzed using Image J software (National Institutes of Health, Bethesda, MD).

Anti-APOL1 (1:2500 HPA018885, Sigma, St. Louis, MO, USA); Anti-APOL1 5.17D12 (1 µg/mL, kindly provided by Genentech, San Francisco, CA, USA); Anti-APOL1 oligoclonal 3.7D6/3.1C1 (0.05 µg/mL, kindly provided by Genentech); Anti-Tubulin (1:5000 T5168, Sigma, St. Louis, MO, USA); Anti-LC3 (1:1000, PM036, MBL, Chicago, MA, USA); Anti-Calreticulin (1:1000, 12238, Cell Signaling, Danvers, MA, USA); anti-Cytochrome C (1:1000, sc-13156, Santa Cruz, Dallas, TX, USA); Anti-Calnexin (1:2000, ADI-SPA-860 Enzo, New York, NY, USA); Anti-SDHB (Succinate Dehydrogenase Complex Iron Sulfur Subunit B, 1:2500, LS C497529, LSbio); Anti-rabbit HRP (1:20,000; 111-035-144, Jackson ImmunoResearch, Baltimore, PN, USA); Anti-mouse HRP (1:10,000; 115-035-166, Jackson ImmunoResearch); Alexa Fluor 488 conjugated Donkey anti Rabbit (1:600; 711-545-152, Jackson ImmunoResearch).

Immunoblot analyses were performed as previously described with minor modifications [33 (link)]. Briefly, exosomal total protein was harvested using RIPA buffer including 1x Halt™ Protease- and Phosphatase-Inhibitor-Cocktail (ThermoFisher Scientific, Waltham, MA, USA). Using Bradford quantification, 25 µg of protein was denaturized and separated by a 4–12% gradient Bis-Tris polyacrylamide gel electrophoresis (Invitrogen^TM, ThermoFisher Scientific). The proteins were then transferred onto a nitrocellulose blotting membrane (Amersham^TM Protran^TM, Amershan, UK) using a wet blotting approach. After blocking for 1 hour at RT, the blots were incubated with following primary antibodies: anti-CD9 (ERP2949) (Abcam), anti-CD63 (H-193) (Santa Cruz), anti-CD81 (NBP2-20564) (Novus Biologicals, Centennial, CO, USA), anti-TSG101 (4A10) (Abcam), anti-GPC-3 (AF2119, R&D Systems) and anti-calreticulin (Cell Signaling, Danvers, MA, USA; Cat. Nr. 2891) overnight at 4 °C on an orbital shaker. The next day, the blots were washed with 1 x PBS/0.05% Tween for at least 30 min, followed by the incubation with horseradish peroxidase (HRP) coupled secondary antibodies (Cell Signaling, anti-mouse Cat. Nr. 7076, anti-rabbit Cat. Nr. 7074) for 1 hour at RT. The blots were washed again and developed with Immobilon Western chemiluminescent HRP substrate using a G:Box XT4 imager (Syngene, Cambridge, UK).

Antibodies against Sig1R(L20) (1:250, Cat# sc‐16203), CYPOR(H‐300) (1:250, Cat# sc‐13984) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Anti‐IP₃R3 (1:1,000, Cat# 610313) antibody was obtained from BD biosciences (San Jose, CA). Anti‐human SOD1 antibody was raised and purified in our laboratory (Bruijn et al, 1997). We also purchased the following commercially available antibodies: anti‐IP₃R1 (L24/18) (1:500, Cat# 817701) (BioLegend, San Diego, CA), anti‐FLAG M2 (1:1,000, Cat# F3165) (Sigma‐Aldrich, St. Louis, MO), anti‐c‐myc (1:400, Cat# 11 667 149 001), anti‐influenza hemagglutinin (HA) (1:1,000, Cat# 11 867 423 001) (both from Roche, Basel, Switzerland), anti‐histone H3 (1:1,000, Cat# 9715), anti‐calreticulin (1:500, Cat# 2891), anti‐PDI (1:1,000, Cat# 2446), anti‐VDAC (1:1,000, Cat# 4661) (Cell Signaling Technology, Danvers, MA), anti‐βIII‐tubulin (1:5,000, Cat# PRB‐435P) (Covance, Princeton, NJ), and anti‐spectrin α II (1:500, Cat# MB1622) (EMD Millipore, Billerica, MA). Alexa‐conjugated secondary antibodies (1:1,000) were purchased from Life Technologies (Grand Island, NY), and horseradish peroxidase (HRP)‐conjugated secondary antibodies (1:5,000) were from GE Healthcare (Waukesha, WI).