Hybond c extra

Ten microliters of input or pull-down sample was run on NuPAGE® Bis-Tris Mini Gel. The gels were run in NuPAGE® MOPS SDS Running buffer at 150 V for ∼1.5 h. The proteins were then transferred to a nitrocellulose membrane (HybondC extra, GE Healthcare) by blotting using 400 mA for 70 min in blotting buffer (NuPAGE® MOPS SDS Running buffer, 20% v/v methanol). The membranes were then blocked with 5% non-fat milk at room temperature for 2 h. After blocking, the membranes were incubated overnight in 2.5% non-fat milk with anti-HA-peroxidase antibody (3F10, Roche) 1 : 1500 diluted, at 4°C in a cold room. The next day, the membranes were washed 2 times for 10 min with PBS-T before being layered with Western Bright TMQuantum and Western Bright TM Peroxide for visualization in a LAS 400 mini machine. All original Western blots are shown in Supplementary document S1.

yCM- and aCM-derived EVs were lysed for 30 min at 4 °C in RIPA buffer (20 nM Tris-HCl, 150 nM NaCl, 1% deoxycholate, 0.1% SDS, 1% Triton X-100, pH 7.8), supplemented with protease and phosphatase inhibitors cocktail (Sigma-Aldrich, Milan, Italy). Protein concentrations were quantified using a BCA Protein Assay Kit. A total of 10 μg of proteins were loaded, electrophoresed on precast 4–20% polyacrylamide Mini-PROTEAN TGX gels (#4561093, Bio-Rad Laboratories, Milan, Italy) at 200V for 35 min, transferred onto nitrocellulose membranes (Hybond-C Extra, GE Healthcare Life Sciences, Milan, Italy), and probed with primary antibodies for CD9 (1:250, #555370, BD Biosciences, Milan, Italy), CD63 (1:1000, #ab271286, Abcam, Cambridge, UK), CD81 (1:1000, #ab79559, Abcam, Cambridge, UK) and beta Actin (1:5000, #ab6276, Abcam, Cambridge, UK). Protein bands were visualized using the WesternBreeze chemiluminescent kit, and densitometric analysis was performed using the ImageJ software (ImageJ software version 1.53j, https://imagej.nih.gov/ij/notes.html, accessed on 1 May 2023).

For total cell protein extraction and western blot analysis, cells were lysed in a buffer containing 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1% Triton X-100, 0.5% deoxycholic acid, 0.1% sodium dodecyl sulfate, Complete protease inhibitor cocktail (Roche) and cleared by centrifugation. The protein concentration was determined by Bradford assay (Bio-Rad). Proteins were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to a nitrocellulose membrane (Hybond-C Extra, GE Healthcare). Membranes were blocked with 5% milk proteins in TBST (50 mM Tris-HCl pH 7.6, 150 mM NaCl, 0.05% Tween-20), and probed with primary antibodies overnight. Membranes were then washed three times with TBST (15 min each) and probed with a HRP-conjugated secondary anti-rabbit antibody for 1 h. After three more washes, signal was detected using HRP conjugated secondary antibodies and ECL (GE Healthcare) and developed on a BioRad Chemidoc MP machine.

Immunoblotting was carried out according to Huang et al. (2007) (link) and Guccione et al. (2010) (link) with slight modifications: cell-free extracts or periplasmic samples were denatured by boiling in Laemmli sample buffer and separated by SDS-PAGE on 8 % (w/v) acrylamide gels and electroblotted onto nitrocellulose membrane (Hybond-C extra; GE Healthcare). Blocking was carried out with Gelatin-NET at room temperature for 1 h. A buffer containing 0.2 % (w/v) BSA and 2 % (w/v) polyvinylpyrolidone (PVP; Mw 24 000– 40 000) in PBS/Tween-20 (PBST) was used for membrane washing and in the dilution of anti-MfrA (Guccione et al., 2010 (link)), anti-GroEL and horseradish peroxidase (HRP)-conjugated secondary antibodies (Sigma). The HRP-conjugated bound antibodies were detected using the enhanced chemiluminesence (ECL) kit from GE Healthcare.

The purified rPbHsp60 and rgp43 were applied to a 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) using a Mini Protean Tetra (Bio-Rad, USA). The gel protein bands were stained with Coomassie Brilliant Blue G-250 (Sigma-Aldrich, USA) or transferred to nitrocellulose membranes (Hybond-C Extra, GE Healthcare, USA). Proteins with known molecular weights were used as standards (LMW-SDS Marker Kit; GE Healthcare). Membranes containing 1 µg of rPbHsp60 or 1 µg of rgp43 were blocked with 3% gelatin in Tris-buffered saline (20 mM Tris-HCl, 150 mM NaCl, pH 7.2) containing 0.05% of Tween-20 (TBS-T), overnight at 4°C. Then, these membranes were washed three times with TBS-T and reacted with serum samples from patients with PCM or with serum samples from NC diluted 1:200 in a solution of 1% gelatin in TBS-T, for 2 h at 37°C. After the incubation, the membranes were washed five times and incubated with alkaline phosphatase-conjugated goat anti-human immunoglobulin antibody (isotyped M, G and A – whole molecule; Merck Millipore, USA), diluted 1:10,000 in a solution of 1% gelatin in TBS-T, for 1.5 hours at 37°C. The reaction was developed with BCIP-NBT (5-bromo-4-chloro-3-indolyl phosphate and nitroblue tetrazolium; Sigma-Aldrich), according to the manufacturer's instructions.

Phosphatidylinositol lipids (200 pmol/µl) suspended in 2:1:0.8 methanol:chloroform:water (Echelon Bioscience, France) were spotted onto nitrocellulose membranes (Hybond C Extra, GE Healthcare). The membranes were dried for 1 h at room temperature and blocked for 1 h in buffer E (50 mM Tris, pH 8, 150 mM NaCl, 0.1% ovalbumin, and 0.01% Tween 20). Freshly purified protein was diluted in buffer E to a concentration of 500 µM and incubated overnight with the membranes. The membranes were washed, and classical immunoblots were performed as described above. Rabbit anti-GST (1:1000 dilutions) and HRP goat anti-rabbit (1:5000 dilutions) sera were used.

Whole protein extraction and immunoblotting analysis were performed as previously described [32 (link)]. In detail, T98G cell pellets were resuspended in ice-cold RIPA buffer (150 mM NaCl, 50 mM Tris-HCl pH 8.0, 1 mM Triton X-100, all from Sigma) supplemented with a Complete Mini protease inhibitor cocktail (Roche, Basel, Switzerland). Protein samples were quantified by Qubit fluorimeter, using a Protein Assay kit (Invitrogen) following the manufacturer’s instructions. Before loading in SDS-PAGE, protein extracts were boiled in Laemmli sample buffer (2% SDS, 6% glycerol, 150 mM B-mercaptoethanol, 0.02% bromophenol blue, and 62.5 mM Tris-HCl pH 6.8). After electrophoresis, proteins were transferred onto a nitro-cellulose membrane Hybond-C Extra (GE Healthcare, Milan, Italy). Membranes were blocked with 5% nonfat milk in PBS containing 0.1% Tween 20 (v/v) and incubated overnight at 4 °C with primary antibodies. The primary employed antibodies were caspase-3 (#D3R6Y) and PARP-1 (#46D11) (Cell Signaling, Danvers, MA, USA; diluted 1:2000); α-tubulin (Cell Signaling, #4967 diluted 1:6000) was used as the internal loading control. Species-specific peroxidase-labeled ECL secondary antibodies (Cell Signaling, diluted 1:4000) were employed. Protein signals were revealed by the Weststar Supernova Kit (Cyanagen, Bologna, Italy) and visualized using Chemidoc MP system (Biorad).