Supersignal west femto maximum sensitivity substrate ecl

Exosomes and cell lysates were spotted on a nitrocellulose membrane (Thermo Fisher Scientific, UK) (0.5 µg protein in 40 µL for all samples – 10 µL at a time, dried under a nitrogen stream before addition of the next 10 µL on the same spot). The membrane was blocked with 3% milk (w/v) prepared in TBS-T (TBS pH 7.6 containing 0.1% Tween-20) for 1 h at RT. The membrane was then incubated with primary rabbit anti-human/mouse antibodies: anti-Alix (monoclonal, clone 3A9, ab117600, Abcam, UK), anti-TSG101 (polyclonal, 14497-1-AP, ProteinTech, UK), anti-CANX (polyclonal, 10427-2-AP, ProteinTech, UK) and anti-GAPDH (monoclonal, clone 14C10, #2118, Cell Signalling Technology, UK) antibodies (1:1000 in 3% milk), overnight at 4°C. The membrane was washed three times with TBS-T, followed by incubation with goat anti-rabbit secondary antibody (polyclonal, ab6721, 1:1000 in 3% milk) for 1 h at RT. The membrane was then washed again as above, and SuperSignal™ West Femto Maximum Sensitivity ECL substrate (Thermo Fisher Scientific, UK) was added to the membrane (50 µL per sample spot). The membrane was incubated with the substrate for 2 min at RT, and then imaged using the Gel Doc™ system (Bio-Rad, USA) under the “Intense Bands” setting. The image obtained was analysed using the Image Lab™ software (Bio-Rad, USA).

Following transfection cells were washed with HBSS and lysed in either 200 μl RIPA buffer or 300 μl 2x Laemmli buffer (62.5 mM Tris-HCl, pH 6.8; 25% glycerol; 2% SDS, 0.01% bromophenol blue, and 5 mM DTT). The total cell lysate (20 μl) was heated to 95°C for 2 minutes and loaded onto a 4–20% Mini-Protean TGX precast SDS-PAGE gel (Bio-Rad) and subjected to electrophoresis at 110V for 60 minutes. Proteins were transferred to PVDF membranes (GE Healthcare) in transfer buffer (25 mM Tris, 192 mM glycine, 20% v/v methanol) for 2 hours at room temperature. Membranes were blocked with 3% w/v non-fat dry milk in Tris-buffered saline (TBS) and incubated with specific primary antibodies for 1 hour at room temperature. Following washing with TBS containing 0.05% Tween-20 (TBS-T) membranes were incubated with peroxidase-linked secondary antibody and developed with SuperSignal West Femto Maximum Sensitivity ECL Substrate (ThermoScientific) and visualized using an imaging system (UVP) with Labworks 4.1.

BEAS-2B cells were lysed in a buffer containing 20 mM Tris, 150 mM NaCl, 1% [vol/vol] Nonidet P-40, 1 mM DTT, 1% [vol/vol] Protease Inhibitor Cocktail, 1% [vol/vol] Phosphatase Inhibitor Cocktail. Total protein content was determined by the Bio-Rad DC Protein Assay kit (Bio-Rad, Hercules, CA), according to manufacturer’s instructions. 20 μg protein for WCL or 15 μL of concentrated lavage or cell culture supernatants was loaded onto polyacrylamide gels. After transfer of proteins to a nitrocellulose membrane, primary antibodies against thioredoxin (ab16965, Abcam -kindly gifted by Dr. Haendeler) and caspase-1 (sc-56036, Santa Cruz) were applied at a dilution of 1:1500 and 1:300 respectively, followed by HRP conjugated secondary antibodies. SuperSignal west femto maximum sensitivity ECL Substrate was used to visualize the proteins of interest (Thermo Scientific) and images were taken on the AIDA image analyzer.