Amersham protran 0.45 m nc

Phos-tag SDS–PAGE was carried out as described previously [18 (link)]. Samples were supplemented with 10 mM MnCl₂ before loading. Gels for Phos-tag SDS–PAGE consisted of a stacking gel [4% (by mass) acrylamide, 125 mM Tris–HCl, pH 6.8, 0.1% (by mass) SDS, 0.2% (by vol) N,N,N′,N′-tetramethylethylenediamine (TEMED) and 0.08% (by mass) ammonium persulfate (APS)] and a separating gel [12% (by mass) acrylamide, 375 mM Tris–HCl, pH 8.8, 0.1% (by mass) SDS, 75 mM Phos-tag acrylamide, 150 mM MnCl₂, 0.1% (by vol) TEMED and 0.05% (by mass) APS]. An amount of 10–30 µg of samples were loaded and electrophoresed at 70 V for 30 min and at 120 V for 2 h in running buffer [25 mM Tris–HCl, 192 mM glycine and 0.1% (by mass) SDS]. Gels were washed in transfer buffer containing 10 mM EDTA and 0.05% (by mass) SDS three times for 10 min, followed by one wash in transfer buffer containing 0.05% (by mass) SDS for 10 min. Proteins were transferred onto nitrocellulose membranes (Amersham Protran 0.45 µm NC; GE Healthcare) at 100 V for 180 min on ice in the transfer buffer without SDS/EDTA. Subsequent immunoblotting was performed as previously described.

Cells were lysed in IPH buffer (50 mM Tris-Cl, 0.5% NP-40%, 50 mM EDTA). Protein lysates were run on 10% polyacrylamide SDS-PAGE gels and transferred to nitrocellulose membranes (Amersham Protran 0.45 µm NC - GE Healthcare Life Sciences). Membranes were blocked for 30 min in 5% milk in TBST buffer and incubated overnight with primary antibody (1:1000). Secondary antibody (1:5000) incubation was carried out for 1 hour after washing with TBST, and before washing and incubation with SuperSignal™ West Pico PLUS Chemiluminescent Substrate (ThermoFisher). The following primary antibodies were used: Anti-Phospho-Stat1 (Tyr701) (58D6) antibody (Cell Signaling Technology 14994), Anti-Stat1 (D1K9Y) antibody (Cell Signaling Technology 80916), Anti-Phospho-Stat3 (Tyr705) (D3A7) antibody (Cell Signaling Technology 9145), Anti-Phospho-Stat5 (Tyr694) (D47E7) antibody (Cell Signaling Technology 4322), Anti-Phospho-IκBα (Ser32) (14D4) antibody (Cell Signaling 2859), Anti-Cleaved PARP (Asp214) (D64E10) antibody (Cell Signaling Technology 5625) and Anti-β-actin (AC-74) antibody (GenWay Biotech Inc. GWB-A0AC74). Densitometry was performed with Fiji^{50 (link)}. Uncropped blots are included in Fig. S8.

EVs were lysed in an RIPA buffer (Thermo Scientific, Milan, Italy Pierce RIPA buffer) containing protease inhibitor (Roche, Milan, Italy Protease inhibitor cocktail tablets) and phosphatases inhibitor (Sigma Aldrich Milan, Italy, Phosphatase inhibitor cocktail 3) for 2 h in ice and the protein concentration was determined using the Bradford assay (Bio-Rad, Milan, Italy Protein Assay Dye Reagent Concentrate). Then, 16 μg of protein was separated using 10% SDS-PAGE and blotted onto a nitrocellulose membrane (GE Healthcare, Milan Italy Amersham Protran 0.45 µm NC ). The membranes were stained with a Ponceau S solution (Sigma, P7170-1L) and then washed with Tris-buffered saline containing 0.1% Tween-20 (T-TBS). After blocking with 5% no-fat dry milk in T-TBS, the membranes were incubated at 4 °C overnight with the following primary antibodies: anti-COL1A2 (Abcam, Cambridge, UK ab96723 1:1000), anti-Cytokeratin 2e (KRT2 Abcam, ab170106 1:800), anti-Alix (Santacruz Biotechnology, Texas, USA, 1A12 1:250), anti-Calnexin (Abcam ab10286 1:1000), and anti-TSG101 (Abcam ab83 1:500). Detection was performed using peroxidase-conjugated secondary antibodies using the ultra-enhanced chemiluminescence system (Thermo Scientific, Super Signal West Femto Maximum Sensitivity Substrate #34095).