Western blotting detection reagent kit

EVs and PC-3 cells (used as a positive control) were lysed in RIPA buffer (150 ml NaCl, 1% Triton X-100, 0.5% Na deoxycholate, 0.1% SDS, 50 ml Tris) and the protein concentration was assessed using Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific, USA) following manufacturer’s instructions. Thirty micrograms of EV and cell proteins were mixed with Laemmli buffer under reduction conditions, denatured for 5 min at 100 °C and loaded on 10% SDS-PAGE gel. Proteins were electroblotted to nitrocellulose membranes and the membranes were blocked with 10% (w/v) fat-free milk and then incubated with the following primary antibodies: anti-TSG101 (Abcam, # ab125011), Calnexin (Abcam, # ab22595), CD9 (Santa Cruz Biotechnology, # sc-13118) and β-actin (Abcam, # ab8224) in 1:1000 dilution. The blots were washed and incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG F(ab’)2-HRP (1:2000) (Santa Cruz, #sc-3837) or chicken anti-mouse IgG-HRP (1:2000) (Santa Cruz, #sc-2962) secondary antibodies, respectively. Protein expression was visualized using Western Blotting Detection Reagent kit (GE HealthCare Lifesciences, Germany).

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).