Aceglow chemiluminescence substrate

Samples were mixed with Laemmli sample buffer with or without DTT (non-reducing conditions were applied for CD63 and CD81) and denatured for 5 min at 90 °C. Afterwards, 10 μg of exosomes were separated by electrophoresis on 10% SDS-polyacrylamide gel and transferred onto a nitrocellulose membrane (Thermo Fisher Scientific, cat. 88018). The membrane blocking was performed for 2 h in 5% skim milk and 0.1% Tween20 in PBS (for anti-CD9, anti-CD63, anti-TSG101) or 5% BSA and 0.1% Tween in PBS (for anti-CD81). Incubation with the primary antibodies, anti-CD63 (Invitrogen, Waltham, MA, USA, cat. 10628D, 1:1500), anti-CD9 (Santa Cruz Biotechnology, Dallas, TX, USA, cat. Sc-13118, 1:500), anti-CD81 (Biorbyt, Cambridge, U.K., cat. Orb388959, 1:500), anti-TSG101 (Becton Dickinson, Franklin Lakes, NJ, USA, cat. 612697, 1:800), was performed overnight at 4 °C. After washing, HRP-conjugated secondary antibody (IgG goat-anti-mouse, Dianova, Hamburg, Germany, cat. 115-035-003, 1:10,000) was added and incubated for 1 h at RT. According to the manufacturer’s instructions, the chemiluminescent signal was elicited by AceGlow™ Chemiluminescence Substrate (VWR Life Science, Radnor, PA, USA, cat. 730-1511).

Liver and spleen lysates of WT and IFNAR^−/− mice were generated by homogenization of organs in liquid nitrogen and subsequent lysis using RIPA-buffer. Samples were incubated for 5 min at 95°C in non-reducing loading buffer containing 10% SDS, and analyzed on a SDS-PAGE according to the method described by Laemmli (cross linker C = 5%, total bis/acrylamide = 15%) (24 (link)) followed by immunoblotting on nitrocellulose membranes (GE Healthcare, Freiburg, Germany). Detection of IL1R1 was performed using a rabbit polyclonal antibody against IL1R1 (1:1,000, Biozol, Germany) and a donkey-anti-rabbit-HRP secondary antibody (1:7,500, Amersham, Freiburg, Germany). Detection of actin was performed using mouse-anti-actin monoclonal antibody (1:1,000, Millipore, Germany) and a rabbit-anti-mouse-HRP secondary antibody (1:7,500, Thermo Fisher Scientific, Germany). After immunoblotting, samples were visualized with AceGlow chemiluminescence substrate (VWR, Darmstadt, Germany) by a CCD camera (Fusin-FX7 Spectra, Vilber Lourmat, Eberhardzell, Germany).

PMA-differentiated U937 MΦ were washed in ice-cold phosphate buffered saline (PBS) and lysed using RIPA (radioimmuniprecipitation assay) buffer pH 7.5 (Cell Signaling Technology Europa, Leiden, The Netherlands), containing protease/phosphatase inhibitor cocktail (Cell Signaling Technology, Boston, USA). The protein concentrations were determined spectrophotometrically using the Pierce BCA (bicinchoninic acid) Protein Assay (Thermo Scientific, Rockford, USA). Proteins were loaded on NuPAGE® Novex® 4–12% Bis-Tris Gels, pre-cast polyacrylamide gels (Life Technologies GmbH, Darmstadt Germany). Proteins were transferred onto 0.45 μm nitrocellulose membranes [Millipore (Billerica, MA, USA)]. Primary Antibodies (see Additional file 1) were added and incubated overnight at 4 °C in blocking buffer (5% milk). Membranes were incubated with enhanced ECL-anti-goat IgG-POD antibody, ECL-anti-mouse IgG-POD antibody or ECL-anti-rabbit IgG-POD antibody. The peroxidase reaction was visualized by AceGlow chemiluminescence substrate (Peqlab, Erlangen, Germany) and documented by the Fusion-SL Advance™ imaging system (Peqlab) according to the instruction manual. The intensity of the specific western blot bands was quantified using the software ImageJ from the National Institutes of Health (Bethesda, USA).