Ecl western blotting detection

Whole cell extracts of mitochondria, resuspended in XT sample buffer (BioRad) and reducing agent (BioRad), were resolved on Criterion XT 12% Bis–Tris gels (BioRad) in XT Mops running buffer (BioRad). Proteins were transferred to Immuno-Blot polyvinylidene difluoride (BioRad) at 100 V for 1 h. Following transfer, membranes were blocked with 5% (w/v) milk and 0.05% (v/v) Tween-20 in PBS (blocking buffer) for 1 h at room temperature or at 4 °C if longer, with blocking. Following blocking, membranes were briefly washed with PBS with 0.2% (v/v) Tween-20 (PBST) and then incubated with primary antibody in PBST with 0.02% (w/v) sodium azide overnight at 4 °C with blocking. Following three successive 10-min washes with PBST at room temperature, horseradish peroxidase–conjugated secondary antibodies were added and incubated for 45 min at room temperature. The membranes were then washed three times for 10 min with PBST and twice for 10 min with PBS. Immunoreactive bands were visualized using ECL Western Blotting Detection (Amersham) or SuperSignal West Pico PLUS (Pierce). Images were captured with a Fluorchem Q (Cell Biosciences, Inc) or on film. Film was scanned before quantification. Quantitation of bands was performed using ImageJ (http://imagej.nih.gov/ij/) and protein expression values were normalized to loading controls.

Cell pellets were lysed for 30 min in ice-cold extract buffer (50 mM TrisHCl), pH 7.4, 250 mM NaCl, 0.1% Nonidet NP40, 5 mM EDTA, 50 mM NaF and a protease inhibitor cocktail (Sigma-Aldrich). Lysates were cleared by centrifuging at 12,000 rpm for 15 min, and the protein concentration was determined using a Bio-Rad assay kit (Bio-Rad Laboratories S.r.l., Hercules, CA, USA). Cell lysates (50 µg) were resolved on 10–12% SDS-PAGE (polyacrylamide gel electrophoresis) gels. Proteins were then transferred to nitrocellulose membranes (Merck Millipore, Burlington, MA, USA). Immunoblotting was conducted with the following antibodies: rabbit anti-γH2AX Cat#9718 (Cell Signaling, Danver, MA, USA, 1:1000) and mouse anti-ACTIN sc-47778 (Santa Cruz Biotechnology, Dallas, TX, USA, 1:500). The secondary antibodies conjugated with horseradish peroxidase (HRP) goat anti-rabbit Cat#170-6515 and goat anti-mouse Cat#170-6516 were purchased from Bio-Rad Laboratories S.r.l. The HRP substrate (ECL Western Blotting Detection, Amersham-Life Science, Amersham, UK) was added, and the signal was detected with the Odyssey Fc instrument (Li-COR, Lincoln, NE, USA). The uncropped version of the gels are shown in Supplementary Figure S6.

MVs_DG75, MVs_MUC1 and extract of DG75-MUC1 cell line (obtained by freeze and thaw method) (30 μg for sample) were separated on 4–12% SDS-PAGE (95V, 220 mA for 90 min at RT) and blotted onto nitrocellulose transfer membrane (Schleicher und Schuell, DE). Prestained protein ladder (10 μL) by Nippon Genetics Europe GmbH was used. After blocking (5% BSA in PBS), membranes were incubated with anti-MUC1 MoAb Ma552 (1:100, 1 h at RT; Monosan), followed by anti-mouse Fc peroxidase-conjugated antibody (1:20,000; 1 h at RT;Jackson ImmunoResearch, USA). Protein bands were detected with enhanced chemiluminescence reagents (ECL Western Blotting Detection; Amersham Biosciences, UK).