Ab10286

Ligatured carotids first extracted with Chloroform:Methanol, were solubilized in NAOH 0.1N (100μL/carotid). The solution is briefly sonicated and centrifuged at 15,000 x g for 10 minutes. The protein concentration was determined with Bradford assay and 40μg of sample were loaded on SDS-PAGE. The ABCA1 expression (ab7360 from Abcam—dilution 1/1000) was quantified using imageJ software. Calnexin expression was used as a loading control for each sample (ab10286 from Abcam—dilution 1/1000). The different samples were quantified and normalized to the same reference sample loaded each time on the SDS-PAGE.

Total protein was extracted from EVs and BMDC cells diluted in 1X RIPA buffer (Bio-Rad), followed by three rounds of 15-second vortexes between five-minute sonication cycles. Protein concentration was determined using the DC protein assay (Bio-Rad) and the Perkin Elmer Enspire 2300 Multilaber reader. EV or cellular proteins (20 μg) were boiled in 1X reducing Laemmli Sample Buffer (Bio-Rad) for 5 minutes at 95°C. Proteins were run on a Mini-Protean TGX precast gel (any kDa; Bio-Rad) and blotted onto Trans-Blot Mini PVDF membranes (Bio-Rad) using the Trans-Blot Turbo Transfer system (Bio-Rad). After blocking for two hours with 5% nonfat milk/PBST at room temperature, membranes were incubated overnight at 4°C with primary antibodies anti-OVA (1: 4,000; clone 0220–1682G, AO Serotec/Bio-Rad), anti-MHCII (1: 4,000; clone ab180779, Abcam), anti-calnexin (1:1,000; ab10286, Abcam), and anti-actin (1:10,000; Sc1616, Santa Cruz), followed by detection with a donkey anti-rabbit (1: 10,000; GE Healthcare). The bands were visualized using enhanced chemiluminescence buffer (GE Healthcare) and analyzed using the ChemiDoc MP Imaging System and Image Lab software version 4.1 (Bio-Rad), respectively.

EVs were lysed in an RIPA buffer (Thermo Scientific, Milan, Italy Pierce RIPA buffer) containing protease inhibitor (Roche, Milan, Italy Protease inhibitor cocktail tablets) and phosphatases inhibitor (Sigma Aldrich Milan, Italy, Phosphatase inhibitor cocktail 3) for 2 h in ice and the protein concentration was determined using the Bradford assay (Bio-Rad, Milan, Italy Protein Assay Dye Reagent Concentrate). Then, 16 μg of protein was separated using 10% SDS-PAGE and blotted onto a nitrocellulose membrane (GE Healthcare, Milan Italy Amersham Protran 0.45 µm NC ). The membranes were stained with a Ponceau S solution (Sigma, P7170-1L) and then washed with Tris-buffered saline containing 0.1% Tween-20 (T-TBS). After blocking with 5% no-fat dry milk in T-TBS, the membranes were incubated at 4 °C overnight with the following primary antibodies: anti-COL1A2 (Abcam, Cambridge, UK ab96723 1:1000), anti-Cytokeratin 2e (KRT2 Abcam, ab170106 1:800), anti-Alix (Santacruz Biotechnology, Texas, USA, 1A12 1:250), anti-Calnexin (Abcam ab10286 1:1000), and anti-TSG101 (Abcam ab83 1:500). Detection was performed using peroxidase-conjugated secondary antibodies using the ultra-enhanced chemiluminescence system (Thermo Scientific, Super Signal West Femto Maximum Sensitivity Substrate #34095).