Sigmafast protease inhibitors and phosphatase inhibitor cocktail 3 and 2

Total proteins were extracted from isolated EVs by lysing samples with ice-cold RIPA buffer supplemented with SIGMAFAST™ Protease Inhibitors and Phosphatase Inhibitor Cocktail 3 and 2 (all from Sigma). Proteins were boiled with Laemmli SDS sample buffer 6X (VWR International), separated on 4–20% MiniPROTEAN®TGX™ Precast Gel, and transferred onto a PVDF membrane with a semi-dry transfer system (all from Bio-Rad Europe, Basel, Switzerland). Membranes were incubated with ALIX (Abcam ab186429; 1:1000), TSG101 (Abcam ab125011, 1:1000), and Syntenin-1 (Abcam ab19903; 1:1000) and Apolipoprotein A1 (APOA1) (Invitrogen 701239, 1:500) primary antibodies. Secondary antibodies IRDye® 680RD or 800CW goat anti-mouse or goat anti-rabbit (LI-COR Biosciences) were used for detection. The infrared signal was detected using Odyssey CLx Detection System (LI-COR Biosciences).

Total proteins were extracted by lysing samples with ice-cold RIPA buffer supplemented with SIGMAFAST™ Protease Inhibitors and Phosphatase Inhibitor Cocktail 3 and 2 (all from Sigma, St. Louis, MI, USA). Protein concentration was determined using BCA (Thermo Fisher Scientific). Proteins were boiled with Laemmli SDS sample buffer 6x (VWR International, Dietikon, Switzerland), separated on 4–20% Mini-PROTEAN®TGX™ Precast Gel, and transferred onto a PVDF membrane with a semi-dry transfer system (all from Bio-Rad Europe, Basel, Switzerland). Membranes were incubated with appropriate antibodies (TF-CD142 ab252918; CD81 ab109201; Syntenin-1 ab19903; TSG101 ab125011; Apo-B48 ab20737; Apo-A1 ab33470 all from aBCAm) and then with IRDye® 680RD or 800CW goat anti-mouse or goat anti-rabbit secondary Ab (LI-COR Biosciences, Lincoln, Nebraska, USA). Infrared signal was detected using Odyssey CLx Detection System (LI-COR Biosciences). Full images of membranes are provided in Fig. S1b.