Anti tsg101

Samples were mixed with Laemmli sample buffer with or without DTT (non-reducing conditions were applied for CD63 and CD81) and denatured for 5 min at 90 °C. Afterwards, 10 μg of exosomes were separated by electrophoresis on 10% SDS-polyacrylamide gel and transferred onto a nitrocellulose membrane (Thermo Fisher Scientific, cat. 88018). The membrane blocking was performed for 2 h in 5% skim milk and 0.1% Tween20 in PBS (for anti-CD9, anti-CD63, anti-TSG101) or 5% BSA and 0.1% Tween in PBS (for anti-CD81). Incubation with the primary antibodies, anti-CD63 (Invitrogen, Waltham, MA, USA, cat. 10628D, 1:1500), anti-CD9 (Santa Cruz Biotechnology, Dallas, TX, USA, cat. Sc-13118, 1:500), anti-CD81 (Biorbyt, Cambridge, U.K., cat. Orb388959, 1:500), anti-TSG101 (Becton Dickinson, Franklin Lakes, NJ, USA, cat. 612697, 1:800), was performed overnight at 4 °C. After washing, HRP-conjugated secondary antibody (IgG goat-anti-mouse, Dianova, Hamburg, Germany, cat. 115-035-003, 1:10,000) was added and incubated for 1 h at RT. According to the manufacturer’s instructions, the chemiluminescent signal was elicited by AceGlow™ Chemiluminescence Substrate (VWR Life Science, Radnor, PA, USA, cat. 730-1511).

Small EVs (total load) were isolated from the serum of patients with rectal cancer by the size exclusion chromatography (SEC) method adopted from Smolarz et al. [28 (link)] and Ludwig et al. [29 (link)] and optimized in our laboratory for MS-based analyses. The size and morphology of vesicles were evaluated by the dynamic light scattering (DLS) using the Zetasizer Nano-ZS90 instrument (Malvern, UK) and by transmission electron microscopy (TEM) using FEI Tecnai Spirit G2 BioTWIN at 120 kV acceleration, according to Thery et al. [30 (link)]. Known exosomal proteins, CD9, CD63, CD81, ALIX, TSG101 (primary antibodies: anti-CD63: (Thermo Fisher Scientific, Waltham, MA, USA ), 10628D, 1:1500; anti-CD9: (Santa Cruz Biotechnology, Dallas, TX, USA ), sc-13118, 1:500; anti-CD81: (Biorbyt, Cambridge, UK), orb388959, 1:500; anti-TSG101: (Becton Dickinson, Franklin Lakes, NJ, USA), 612697, 1:800; anti-Alix (Cell Signaling Technology, Danvers, MA, USA), 2171S, 1:1000, 2171S, 1:1000) were analyzed in sEV fraction by Western blot technique. The concentration of isolated PKH67-labeled sEV fraction was measured by Amnis ImageStream®X Mark II (Luminex, Seattle, WA, USA) flow cytometer. The concentration of proteins in the analyzed fraction was determined by the BCA method (PierceTM BCA Protein Assay kit, Thermo Fisher Scientific, Waltham, MA, USA), according to the manufacturer’s instructions.

EV sample was concentrated using Concentrator plus 5305 Vacuum Centrifuge (Eppendorf AG, Germany), and protein concentration was measured with Pierce BCA Protein Assay Kit (Thermo Scientific). Concentrated sEV preparations and lysate of HCT116 cells were lysed in Pierce Lane Marker Reducing Sample Buffer (Thermo Scientific), heated for 5 min at 95 °C, and subjected to electrophoresis using 10% SDS-PAGE. Proteins were transferred to an Immobilon-P PVDF Membrane (Merck Millipore) and the excess protein binding sites on the membrane were saturated with 5% bovine serum albumin blocking buffer (1 × TBS, 0.1% Tween-20) for 1 h. The membrane was incubated overnight at 4 °C with primary antibody. The following antibodies were used: anti-CD81 (1:250, mouse, catalogue number sc166029), anti-CD63 (1:300, mouse, sc5275) from Santa Cruz Biotechnology, anti-Alix (1:20000, rabbit, ab186429) from Abcam, anti-TSG101 (1:200, mouse, 612696) from BD Biosciences, and anti-Calnexin (1:1000, rabbit, 2679) from Cell Signaling. After incubation, the membrane was washed three times with 5% TBS-Tween and then, incubated with peroxidase-labelled secondary antibody (Santa Cruz Biotechnology) for one hour. After three washes, immobilized proteins were detected utilizing Clarity Western ECL Substrate (Bio-Rad) and the UVITEC chemiluminescence imager (UVITEC Cambridge, UK).