Mouse anti a33

sMV/exosome preparations (10 μg protein) were lysed in SDS sample buffer, resolved by SDS-PAGE, electrotransferred as previously described11 (link)79 . Membranes were probed with primary mouse anti-CD9 (1:1000, BD Biosciences), mouse anti-Alix (1:1000, Cell Signaling), and mouse anti-A33 (1 μg/ml, a kind gift from Dr. A. Scott, Ludwig, Austin Campus). Membranes were further incubated with secondary antibodies horse radish peroxidase (HRP)-conjugated anti-mouse IgG (1: 15,000, Sigma) and IRDye 800 goat anti-mouse IgG (1: 15000, Li-COR Biosciences). All antibody incubations were carried out using gentle orbital shaking at RT. Proteins were visualised by incubating membranes with Western HRP substrate (Merck-Millipore) followed by imaging with ChemiDoc MP System (Bio-Rad) or imaged directly with the Odyssey Infrared Imaging System, version 3.0 (LI-COR Biosciences, Nebraska USA).

Sample protein concentrations were determined using one-dimensional SDS-PAGE/SYPRO Ruby protein staining/densitometry, as described [5] (link), [6] (link). Briefly, samples were lysed in SDS sample buffer for 20 min at room temperature, and proteins (10 µg/sample) resolved by SDS-PAGE, electrotransferred onto nitrocellulose membranes using the iBlot Dry Blotting System (Life Technologies), and membranes blocked with 5% (w/v) skim milk powder in Tris-buffered saline containing 0.05% (v/v) Tween-20 (TTBS) for 30 min. Membranes were probed overnight in TTBS with primary mouse anti-CD9 (at 1∶1000) and mouse anti-TSG101 (at 1∶1,000) from BD Biosciences, mouse anti-Alix (at 1∶1,000) from Cell Signalling, or mouse anti-A33 (1 µg/ml) (gift from Dr A Scott, Ludwig Institute for Cancer Research, Melbourne). After washing with TTBS (3×10 min) membranes were incubated with horse radish peroxidase (HRP)-conjugated anti-mouse IgG (Sigma) or IRDye 800 anti-mouse IgG (Li-COR Biosciences). Proteins were visualized by incubating membranes with Western HRP substrate (Merck-Millipore) followed by imaging with ChemiDocMP System (Bio-Rad) or by imaging directly with the Odyssey Infrared Imaging System (v3).