Anti tsg101

Density gradient fractions were loaded on a 4–20% gradient Tris-Glycine precast polyacrylamide gel (Bio-Rad, Hercules, CA), separated by standard SDS-PAGE procedures and transferred onto PVDF membranes. (Immobilon, EMD Millipore, Billerica, MA). Primary antibodies used in this study were: anti-CD63 (1:1,000, #ab217345, Abcam, Cambridge, UK), anti-Alix (1:1,000, #ABC40, EMD Millipore), anti-TSG101 (1:1,000, #PA5-31260, ThermoFisher Scientific), anti-Rab35 (1:1,000, #9690, Cell Signaling Technology, Danvers, MA), anti-Rab5B (1:5,000, #sc-598, Santa Cruz Biotechnology, Dallas, TX), anti-Rab7 (1:5,000, #R8779, Sigma-Aldrich) and anti-EEA1 (1:1,000, #07-1820, EMD Millipore). The secondary antibodies used were HRP-conjugated anti-rabbit and anti-mouse antibodies from Jackson ImmunoResearch (West Grove, PA, US). The membranes were incubated in chemiluminescent fluid (Pierce, Rockford, IL, US) for 5 min, and chemiluminescence was visualized on Reflection Autoradiography films. Ponceau staining (Sigma-Aldrich) was used as a loading control. Protein bands were quantified through the open source software ImageJ (National Institute of Health (NIH), Bethesda, MD, US). Data are shown as the densitometric ratio between Ts2 and 2N controls, after normalization for protein concentration of each fraction.

In preparation for Western blots, exosome fraction #4 was concentrated by centrifugation on a 100 K Amicon Ultra 0.5 mL centrifugal filter (EMD Millipore, Billerica, MA, USA) at 5000 × g. Western blots were performed as previously described [12 (link)]. PVD membranes were incubated overnight at 4 °C with various antibodies (Abs) as indicated below. Exosomes (10 μg protein) were loaded in each lane and tested for the presence of exosome markers, including markers related to hematopoiesis, leukemia blasts markers, and Leukemia Associated Antigens (LAAs) as previously described [12 (link)]. The following Abs were used: anti-TGF-β1 (Cell Signaling, #3711, 1:1000); anti-TNF-a (Cell Signaling, 3707S, 1:1000); anti-TSG101 (Thermo Fisher, PA5–31260, 1:500); anti-EPOR (R&D, MAB307, 1:500); anti-CD26 (R&D, AF1180, 1:500); anti-CLL-1 (R&D, AF2946, 1:2000); anti-CD34 (Santa Cruz, sc-7045, 1:500); and anti-CD33 (Thermo Fisher, WM53, 1:500). Band intensities on exposed films were quantified using Image J software (NIH, USA). The examined band intensity was normalized to the intensity of TSG101 used as a marker of the exosome endocytic origin. The integrated pixel value was determined for each protein band by multiplying image intensity and band area after subtracting the mean background value.

Total proteins were extracted from EV pellets with RIPA Lysis and Extraction Buffer (Thermo Fisher Scientific, Waltham, MA). 10 μg of proteins were loaded on a 4–20% precast polyacrylamide gel (Bio-Rad Laboratories, Hercules, CA) and separated by SDS-PAGE, then transferred to PVDF membranes. Proteins were detected with anti-CD63, anti-Rab27A, anti-TSG101 and anti-Calnexin (Thermo Fisher Scientific), followed by species specific horse-radish peroxidase labeled antibodies (Bio-Rad) and signal detection by V3 Western Workflow™ (Bio-Rad).

Protein lysates were collected using 1x RIPA lysis buffer (Millipore) and concentrations were determined using the Pierce BCA assay kit (Thermo Scientific). Proteins (1μg) were separated on 12% MINI-PROTEAN TGX pre-cast gels (Bio-Rad) and transferred onto PVDF membranes using the Trans-Blot Turbo Transfer System (Bio-Rad). Membranes were blocked in 5% milk in TBS-T for 1 hour and then incubated with primary antibodies in 5% milk in TBS-T overnight at 4°C. Primary antibodies were used at the following concentrations: anti-TSG101 (Invitrogen) at 1:1000, anti-CD81 (Santa Cruz Biotechnology) at 1:1000, and anti-Histone H3 (Abcam) at 1:5000. The next day, blots were washed with 1X TBS-T 3 times, then incubated with secondary antibodies in 5% milk in TBS-T at room temperature for 45 minutes. The following secondary antibodies were used at a concentration of 1:10,000: anti-Mouse ECL (GE Healthcare) and anti-Rabbit IgG (Cell Signaling). Blots were washed with 1X TBS-T three times and then treated with SuperSignal™ West Fenmto Maximum Sensitivity Substrate (Thermo Scientific) to visualize bands.