Mini protean tris glycine extended gels

Western blotting was performed as previously described^{30 (link)}. Total protein from cells (10 μg) and exosomes (10 μg) was fractionated using an electrophoretic gradient across Mini-PROTEAN tris-glycine extended gels (BIO-RAD, Richmond, CA, USA). Measurement of protein concentration was performed using a Bradford Protein Assay Kit (Takara Bio), according to the manufacturer’s instructions. The gels were then transferred onto Immun-Blot PVDF membranes (BIO-RAD) under wet electrophoretic conditions. The blotted protein was blocked for 1 hr at room temperature with Odyssey blocking buffer in PBS (LI-COR, Lincoln, NE, USA) and was followed by incubation overnight at 4 °C with the following primary antibodies: 1:1000 anti-CD63 mouse monoclonal antibody (BD Biosciences, San Jose, CA, USA); 1:1000 anti-cytochrome-c mouse monoclonal antibody (BD Biosciences); 1:1000 anti-calnexin rabbit monoclonal antibody (Abcam) and 1:1000 hFAB Rhodamine (#12004168 - Bio-Rad). Primary antibodies were detected by IRDye 800CW anti-rabbit IgG and IRDye 680RD anti-mouse IgG secondary antibodies (LI-COR) and were incubated with the protein-blotted membrane for 1 hr at room temperature. Fluorescence was then detected on the Odyssey imaging system (LI-COR).

Total protein from cells (10 μg) or EVs (30 μg) was fractionated using an electrophoretic gradient across Mini-PROTEAN^® tris-glycine extended gels (BIO-RAD, Richmond, CA, USA). Loading samples were normalized according to the protein concentrations quantified using the Bradford assay. The gels were then transferred onto the Immun-Blot^® PVDF membrane (BIO-RAD) under wet electrophoretic conditions. The blotted protein was blocked for 1 h at room temperature with Odyssey^® blocking buffer in PBS (LI-COR, Lincoln, NE, USA) followed by incubation overnight at 4 °C with the following primary antibodies: 1:200 anti-MCT1 mouse monoclonal antibody (sc-365501; Santa Cruz Biotechnology); 1:200 anti-CD81 mouse monoclonal antibody (sc-23962; Santa Cruz Biotechnology); 1:2000 anti-tubulin hFAB Rhodamine (BIO-RAD). Thereafter, IRDye^® 800CW anti-mouse IgG secondary antibodies (LI-COR) were incubated with the protein-blotted membrane for 45 min at room temperature. Fluorescence was detected using the Odyssey^® imaging system (LI-COR).

Immunoprecipitation (IP) for analytical separation of Ago2 from patient serum samples was performed using Protein G Sepharose 4 Fast Flow^® (GE Healthcare, Amersham, UK) with anti-Ago2 monoclonal IgG antibody (Wako, Osaka, Japan) according to the product manual. Total protein from cells (10 µg) and exosomes (1 µg) was fractionated using an electrophoretic gradient across Mini-PROTEAN^® tris-glycine extended gels (BIO-RAD, Richmond, CA, USA). Loading samples were normalized according to protein concentrations quantified using the Bradford assay^{45 (link)}. The gels were then transferred onto the Immun-Blot^® PVDF membrane (BIO-RAD) under wet electrophoretic conditions. The blotted protein was blocked for 1 hr at room temperature with Odyssey^® blocking buffer in PBS (LI-COR, Lincoln, NE, USA) and was followed by incubation overnight at 4 °C with the following primary antibodies: 1:1000 anti-CD9 mouse monoclonal antibody (Abcam, Cambridge, MA, USA); 1:1000 anti-cytochrome-c mouse monoclonal antibody (Abcam); and 1:10000 anti-β-actin mouse monoclonal antibody (Sigma-Aldrich, St. Louis, MO, USA). Thereafter, IRDye^® 800CW anti-rabbit IgG and IRDye^® 680RD anti-mouse IgG secondary antibodies (LI-COR) were incubated with the protein-blotted membrane for 1 hr at room temperature. Fluorescence was then detected on the Odyssey^® imaging system (LI-COR).