Trans blot turbo transfert pack

Proteins (20 µg of cellular protein extract and 5 µL of concentrated culture medium) were separated on 10% SDS-PAGE, transferred on 0.22 µm nitrocellulose membranes (Trans-Blot® Turbo^TM Transfert Pack, Bio-rad) and then incubated with antibodies as previously described^{6 (link)}. The primary antibodies used for western blot analysis were: CLU (sc-6419), Bcl2 (sc-493), and GAPDH (sc-365062) antibodies from Santa Cruz Biotechnology; beclin-1 (#3738), LC3B (#2775), and cleaved caspase 3 (#9664) antibodies from Cell signaling; mono-and poly-ubiquitin antibody (BML-PW8810) from Enzo Life Sciences; P62 (610498) from BD Transduction Laboratories; β-actin antibody (A5316) from Sigma-Aldrich and sarcomeric-actin antibody (m0874) from Dako. The horseradish peroxidase-labeled secondary antibodies used were: anti-rabbit IgG (NA934V) and anti-mouse IgG (NA931) antibodies from GE healthcare and anti-goat IgG antibody (sc-2020) from Santa Cruz Biotechnology. The dilution of antibodies used for Western blot is provided for each sample analyzed (Supplementary Table 1). The Chemidoc® camera (Biorad) was used for imaging the membranes and densitometric measurements of the bands were analyzed with the Image Lab software (Bio-Rad).

Soluble proteins (10 to 50 µg) were resolved on NuPAGE 4–12% Bis-Tris Protein Gels (Thermo Fisher Scientific) or on SDS-PAGE gels (12 or 15%, depending on the proteins analyzed) and transferred on 0.2 μm nitrocellulose membranes (Trans-Blot^® TurboTM Transfert Pack, Bio-Rad). Equal total proteins loads were verified by Ponceau red (0.1% Ponceau, Sigma-Aldrich), 5% acetic acid (v/v) staining of the membranes. The membranes were then blocked in 5% milk or 5% BSA in TBS-Tween buffer for 1 h before 4 °C overnight incubation with primary antibodies diluted in blocking solution. Blots were then washed three times with TBS-Tween 0.1% buffer and incubated with corresponding secondary antibodies for 1 h (1/5000 to 1/10,000) in blocking solution. The Chemidoc^® camera (Bio-Rad) was used for imaging and densitometry analysis after membranes were incubated with enhanced chemiluminescence (ECL™) Western blotting detection reagents (GE Healthcare, Chicage, IL, USA).

Proteins were extracted from HeLa cells derived-lEVs and -sEV in RIPA buffer as previously described [40 (link)] and their concentration was measured using Bradford assay (#5,000,006, Bio-Rad Laboratories) according to the manufacturer instructions. Ten µg of proteins were separated on 4–12% SDS-PAGE and transferred on 0.22 µm nitrocellulose membranes (Trans-Blot^® Turbo™ Transfert Pack, Bio-rad). After blocking with 5% non-fat dry milk in TBS-Tween 0.1% buffer for 1 h at room temperature, the membranes were incubated with primary antibodies at a dilution 1:1000 in TBS-Tween 0.1% buffer with 5% non-fat dry milk (Tetraspanin CD81: sc-166029, Santa Cruz Biotechnology; MVP (major volt protein): 16,478-1-AP, Proteintech) or with 5% BSA (Tetraspanin CD9: #13,403, Cell Signalling) at 4 °C with gentle shaking overnight. The membranes were then washed three times for 10 min with TBS-Tween 0.1% buffer and incubated for 1 h with the corresponding horseradish peroxidase-labelled secondary antibodies (anti-rabbit IgG NA934V and anti-mouse IgG NA931, GE healthcare) at a dilution 1:5000 in the blocking solution. After three washes with TBS-Tween 0.1% buffer, the membranes were incubated for 5 min with Clarity™ Western ECL Substrate (Bio-Rad Laboratories). Images were acquired using ChemiDoc Imaging System (Bio-Rad Laboratories).