Sc 7298

Loading buffer was added to 10 µg of EV protein sample and boiled at 95 °C for 5 min, separated in a 12% SDS-polyacrylamide gel (SDS-PAGE) and electrophoretic transferred to nitrocellulose membranes. Membranes were blocked in 2.5% nonfat dry milk in 1x TBS-T (0.5% Tween-20), at room temperature and incubated with the following primary antibodies: rabbit anti-CD9 (1:2000) (sc-9148; Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA), rabbit anti-CD10 (1:3000) (GTX111680; Genetex, Irvine, CA, USA), mouse anti-HSC70 (1:2000) (sc-7298; Santa Cruz Biotechnology, Inc.), mouse anti-TSG101 (1:1000) (sc-7964; Santa Cruz Biotechnology, Inc.). Membranes were then washed with 1x TBS-T (0.5% Tween-20). The secondary antibodies used were the mouse IgGκ BP conjugated to HRP (1:2000) (sc-516102; Santa Cruz Biotechnology, Inc.); and the mouse anti-rabbit IgG-HRP (1:2000) (sc-2357; Santa Cruz Biotechnology, Inc.). Secondary antibodies were incubated for 2 h, with agitation at room temperature. Following TBS-T washes, protein bands were detected using the chemiluminescence reagent WesternBright ECL HRP substrate (Advansta Inc., San Jose, CA, USA.) and images acquired with G:BOX Chemi XX9 gel imaging system (Syngene, Cambridge, UK).

Cells were harvested and lysed with NP40 lysis buffer (Invitrogen). Protein concentration was determined by using the Bio-Rad Dc protein assay kit (Bio-Rad Laboratories). In total, 20–30 μg of protein from each sample was loaded onto 8–12% polyacrylamide gels. The protein was transferred to a nitrocellulose membrane and incubated with 5% milk in phosphate buffered saline with 0.1% Tween-20 (PBS-T), followed by an overnight incubation of primary antibody diluted in 1 in 1000–2000 in 2% milk PBS-T at 4 °C. The blots were then washed three times with PBS-T and incubated with the relevant horseradish peroxidase (HRP)-conjugated antibody diluted 1:1000 in 2% milk in PBS-T, for 1 h at RT. Chemiluminescence was detected by using Mini Chemiluminescent Imaging and Analysis System (Sagecreation). Rabbit mAb against human ROCK1 (CST, 4035, 1:1000) and rabbit mAb against human/mouse ROCK2 (CST, 8236S, 1:1000) were used in this part. Hsc70 was used as loading control (Santa Cruz, sc-7298, 1:1000). Primary antibody details were mentioned in the immunohistochemistry section. For the inhibitor experiment, NPC/TPC were first treated with either placebo, 3-BP, ROCK inhibitor for 24 h before harvesting for western blotting. The uncropped and unprocessed scans of all western blots were given in the Source Data File.

Quantitative analysis of free amino acids in the pig sera was performed at the Proteomics and Metabolomics core facility at IGTP using the Waters’ Pico Tag method. This method involves precolumn derivatization of the amino acids with phenylisothiocyanate and posterior reverse-phase high-performance liquid chromatography separation adapted from ref. 55 (link). Plasma levels of the molecule HSC70 were determined by Western blot using the mouse anti-HSC70 antibody sc-7298 (Santa Cruz Biotechnology) and the Revert Total Protein Stain (LI-COR) as the loading control.

For immunoblotting, culture medium was discarded and cells were washed with PBS and lyzed using lysis buffer: Tris 25 mM pH 7.4, 0.1% SDS, NaCl 150 mM, 1%NP-40 and 0.5% sodium deoxycholate supplemented with protease inhibitors (EDTAfree, Roche, Basel, Switzerland). Samples were then sonicated and their total protein contents were quantified using Biorad protein reagent assay. For immunoblotting, 30 µg of protein was mixed with Laemmli buffer and boiled during 10 min. The samples were next separated on NuPAGE^® Novex^® Bis-Tris 4–12% gels kit (Invitrogen) and transferred to PVDF membranes (Trans-blot^® Turbo™ Transfer System, Biorad, Hercules, CA, USA) before performing immunoblotting using the following primary antibodies against: GFP (anti GFP goat polyclonal antibody, sc-5384, Santa Cruz Biotechnology), CYP2D6 (anti-CYP2D6, 738667S, Cell Signaling), CYP3A4 (anti-CYP3A4, AB1254, Millipore) and HSC70 (anti-HSC, mouse monoclonal antibody, sc-7298, Santa Cruz Biotechnology, Dallas, TX, USA). Primary antibodies were detected using secondary rabbit or mouse antibodies coupled to horseradish peroxidase (HRP) (Dako, Denmark). Detection of the immune complex was performed using a chemiluminescent HRP substrate (Pierce™ ECL Substrate, Waltham, MA, USA) using the Fusion FX system (Vilber-Lourmat, Eberhardzell, Germany).

Protein extraction was performed using the Protein Extraction Buffer Invitrogen (Thermo Fischer Scientific, Waltham, MA, US). Twenty micrograms of protein were run on a 7%, 10%, or 12% SDS-PAGE gel, depending on the size of the protein, and transferred onto a nitrocellulose membrane (GE Healthcare) using a wet-blotting system (Bio Rad Laboratories) for 2 h at 100 V. Non-specific binding was blocked by incubating the membranes in 5% skimmed milk in TBS-Tween for 2 h. The blocked membranes were incubated overnight at 4 °C with the primary antibodies diluted in TBS/BSA 2% against FOXM1 (1:500, sc-502, Santa Cruz), XIAP (1:1000, 2045S, Cell Signaling), c-IAP 1 (1:2000, AF8181, R&D Systems), survivin (1:1000, 2808S, Cell Signaling), Mcl-1 (1:100, #4572, Cell Signaling) and Hsc70 (1:1000, sc-7298, Santa Cruz). After overnight probing with primary antibodies, the membranes were incubated with secondary antibodies anti-mouse (1:40000, GE Healthcare), anti-rabbit (1:40000, GE Healthcare), and anti-goat (1:2000, NB7362, Novus Technologies; Littleton, CO, USA) for 1 h at room temperature. Protein band signals were developed using the Clarity Max™ substrate (Western ECL Substrate-BioRad Laboratories, Hercules, CA, USA) and detected using the C-DiGit blot scanner (Li-cor Biociences, Lincoln, NE, USA).

In Western blot (WB) experiments, 5–15 μg of proteins from CLB-Ga and CLB-Sedp cell lysates were separated by SDS-PAGE and transferred onto a nitrocellulose membranes. The membrane was saturated with a solution of TBS-T (20 mM Tris–HCl, pH 7.0, 130 mM NaCl, 0.1% Tween 20) containing 5% milk, then incubated with various antibodies diluted in TBS-T solution containing 2.5% milk. The anti-casp3 Alexis (1:250) (ALX-804-305-C100, Enzo Life Sciences), anti-Parp1 (1:100) (AM30 Calbiochem), anti-K48-Linkage specific polyubiquitin (1:1000) (8081S, Cell Signaling), anti-HSC70 (1:1000) (sc-7298, Santa Cruz Biotechnology) and anti-Ku80 (1:2000) (ab3715 Abcam) murine monoclonal antibodies were incubated for 1 h at room temperature (RT) with the membrane. The primary antibodies were detected with anti-mouse or anti-rabbit secondary antibodies coupled to HRP.