Ecl system

Equal amounts of EVs (6.8 · 10⁹ particles) were loaded in 10% SDS-PAGE gels, either under reducing or non-reducing conditions, as indicated in the experiments, and transferred to membranes with Trans-Blot® Turbo™ Transfer Packs (Biorad). Membranes were blocked using 5% non-fat dry milk in PBS containing 0.1% Tween 20 (PBS-T). Primary antibody was incubated for 1 h in PBS-T and, after washing, membranes were incubated with the appropriated secondary antibody. Proteins were visualized using the ECL system (Amersham Biosciences). Antibodies used were: monoclonal anti β-actin produced in mouse (Sigma) at 0.13 µg/ml; purified mouse monoclonal anti-CD9 (MEM62), -CD63 (MEM259) and -CD81 (MEM-38) antibodies (kind gifts from Vaclav Horejsi, Croatia); horseradish peroxidase-conjugated goat anti-mouse antibody (Dako). Rabbit polyclonal anti-calreticulin (Novus Biologicals) was used for WB at 4 µg/ml and biotinylated anti-EpCAM (clone VU-1D9) at 1 µg/ml.

Total protein was extracted from cultured cells and quantified using Trizol reagent (Invitrogen) following the protocols. Equal amounts of protein from different cells were separated by 10% SDS-PAGE and transferred to a nitrocellulose membrane (Bio-Rad, Hercules, CA). The membrane was blocked with 5% non-fat milk and incubated with antibodies against MHC-ІІ (ab55152, Abcam), IL-1β (#12242, CST), CD206 (ab64693, Abcam), Arg-1 (#9819, CST), GAPDH (ab8245, Abcam), CEBPA (ab40764, Abcam), and β-actin (ab8245, Abcam) at the concentration of 1:1000 at 4°C overnight. The blots were then incubated with horseradish peroxidase–conjugated secondary antibodies in blocking buffer at room temperature for 2 h. The signal was detected using ECL system (Amersham Pharmacia) and quantified by scanning densitometry and computer-assisted image analyzer.

Prior to electrophoresis, samples were adjusted to 2% (w/v) SDS and 3 mM β-mercaptoethanol and boiled for 10 min. Proteins were resolved by SDS-PAGE with 10% (w/v) resolving gels and 5% (w/v) stacking gels, and PageRuler prestained molecular mass standards (Fermentas). Chemiluminescent western blot analysis was performed using the ECL system (Amersham Biosciences, Pittsburgh, PA, USA) as previously described [19 (link),32 (link)], with modification. Western blots were probed with 1:5000 dilution of the anti-amylase antibody [19 (link)], and then 1:5000 dilution of HRP goat anti-rabbit IgG secondary antibody (Zymed, Rockford, IL, USA). Purified amylase (40 ng) was used as a standard for Western blot analysis. Anti-amylase antibody was pre-adsorped using cell extracts from the amyA disruption strain, PBL2004 [19 (link)], to remove non-specific antibodies, as previously described [37 ] with modification. Pre-wetted nitrocellulose membrane (80 μg protein/cm²) was incubated with sonicated cell pellets for 3 h at room temperature, and blocked overnight in 2.5% (w/v) milk (Nestle, Glendale, CA, USA) in PBS buffer (10 mM NaH₂PO₄ and 150 mM NaCl). Nitrocellulose membranes were washed three times with PBS buffer and incubated with 1:5000 dilution of anti-amylase antibody in 0.5% (w/v) milk in PBS buffer for 2 h at room temperature.