D610 dslr body

EVs were heated for 5 min at 95°C with reducing Laemmli buffer and amounts corresponding to 100 μL plasma were loaded per lane of a 10% SDS-PAGE gel. After separation, the proteins were transferred to nitrocellulose membranes, which were subsequently blocked using 10% (w/v) fat-free milk. Membranes were incubated with primary antibodies against TSG101 (Abcam, #ab15011, 1:1000 dilution), Calnexin (Abcam, #ab22595, 1:2000 dilution) and PDCD6IP/ALIX (Santa Cruz Biotechnology, #sc-166952, 1:1000 dilution) overnight at +4˚C. After washing in TBST, membranes were incubated for 1h at room temperature with anti-rabbit IgG, F(ab’)2-HRP (Santa Cruz Biotechnology, #sc-3837, 1:2000 dilution), goat anti-mouse m-IgG BP-HRP (Santa Cruz Biotechnology, #sc-516102, 1:2000 dilution), or HRP-conjugated antibody against CD63 (Novus Biologicals, #NBP2-34779H, 1:2000 dilution). After washing in TBST, immunoreactive bands were visualized using Amersham™ ECL Select™ Western Blotting Detection Reagent kit (GE HealthCare Lifesciences) and pictures were taken using a Nikon d610 dSLR body (Nikon) with Sigma 35mm f/1.4 DG HSM Art lens (Sigma).

EVs were lysed in RIPA buffer (50 mM Tris, pH 8.0, 150 mL NaCl, 1% Triton X-100, 0.5% Na deoxycholate, 0.1% SDS). Protein concentration was measured using PierceTM BCA Protein Assay Kit (Thermo Fisher Scientific). LNCaP cells (ATCC, Manassas, VA, United States) were used as a positive control. Equal fraction (one fifth) of the EV proteins and 10 µg of cellular proteins were separated by 10% SDS-PAGE, transferred to nitrocellulose membranes and blocked using 10% (w/v) fat-free milk. Membranes were incubated with primary antibodies against TSG101 (Abcam, #ab15011, 1:1000 dilution), Calnexin (Abcam, #ab22595, 1:2000 dilution), CD63 (Santa Cruz Biotechnology, #sc-5275, 1:500 dilution) and PDCD6IP/ALIX (Santa Cruz Biotechnology, # sc-166952, 1:1000 dilution). Membranes were washed and incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG, F(ab’)2-HRP: (Santa Cruz Biotechnology, #sc-3837) or goat anti-mouse m-IgGκ BP-HRP (Santa Cruz Biotechnology, #sc-516102) in 1:2000 dilution. Immunoreactive bands were visualized using Western Blotting Detection Reagent kit (GE Healthcare Lifesciences) and pictures were taking using a Nikon d610 dSLR body (Nikon) with Sigma 35 mm f/1.4 DG HSM Art lens (Sigma).

Urinary EVs were heated for 5 min at 95˚aC with reducing Laemmli buffer and amounts corresponding to 1.5ml urine were loaded per each lane of a 10% SDS-PAGE gel. LNCaP cell lysate, prepared in 1x RIPA and heated with reducing Laemmli prior to loading, was used as a control (10 µg proteins per lane). PageRuler™ Prestained Protein Ladder, 10 to 180 kDa (ThermoFisher Scientific) was loaded onto each gel for assessment of protein molecular weighs. After separation by SDS-PAGE, the proteins were transferred to Amersham Protran Supported 0.45 NC membranes (Merck Millipore), which were subsequently blocked using 10% (w/v) low-fat milk. Membranes were incubated with primary antibodies against TSG101 (Abcam, #ab15011, 1:1000 dilution), Calnexin (Abcam, #ab22595, 1:2000 dilution), or CD63 (Sino Biological, # 11271-T16, 1:300 dilution) overnight at + 4 ˚C. Membranes were washed in TBST and incubated for 1 h at room temperature with anti-rabbit IgG, F(ab’)2-HRP (Santa Cruz, #sc-3837, 1:2000 dilution), or goat anti-mouse m-IgG BP-HRP (Santa Cruz, # sc-516,102, 1:2000 dilution). After washing again in TBST, immunoreactive bands were visualized using Western Blotting Detection Reagent kit (GE HealthCare), and pictures were taken using a Nikon d610 dSLR body (Nikon) with Sigma 35 mm f/1.4 DG HSM Art lens (Sigma).