Ab10895

Proteins were extracted from 1 ml ultracentrifuged plasma (120,000 × g for 2.5 h) using the DE buffer (consisting of 20 mM Tris-HCL, 12 mM 2-mercaptoethanol, 1 mM EGTA, 1 mM EDTA, 1% Triton-X 100, together with 10% glycerol) that contained the protease inhibitor mix (GE Healthcare, Uppsala, Sweden). Total proteins were quantified using Bradford, then 5 µg protein from each sample was separated on a 12% SDS-PAGE. The protein bands were then transferred onto nitrocellulose membranes and blotted using polyclonal antibodies against CD9 (1:1,000 diluted, Abcam ab10895, Cambridge, UK), CD63 (1:1,000 diluted, Abcam ab92726, Cambridge, UK), and Tsg-101 (1:1,000 diluted, Abcam ab30871, Cambridge, UK) at 4°C, overnight. GAPDH was used as a loading control. The target protein expression level was quantified by densitometry analysis using the ImageJ software.

Protein extracts from exosomes or liver tissue were separated by 10% SDA-polyacrylamide gel electrophoresis (PAGE) and electrotransferred to a 0.2 µm nitrocellulose membrane. After blocking for 1 h in bovine serum albumin (BSA), the membranes were incubated with primary antibodies at 4 °C overnight and then incubated with a secondary antibody. After the membranes were washed 3 times with TBS and tween 20 (TBST), the protein bands were visualized using an Odyssey two-color infrared fluorescence imaging system (LI-COR, Nebraska, USA) according to the manufacturer’s protocol and analyzed with a gel image analyzer. The antibodies used in this study were as follows: CD63 (ab10895), CD9 (ab92726, Abcam, Cambridge, UK), CD81 (18250-1-AP, Proteintech), NLRP3 (ab214185), ASC (ab47092), caspase-1 (ab1872) (Abcam, Cambridge, UK) and GAPDH (60004-1-Ig, Proteintech, Rosemont, USA).

Raw264.7-EVs and serum-EVs were isolated and purified as described previously, with added modifications [29 (link)]. Briefly, Raw264.7 culture medium and serum were harvested and centrifuged at 300 g for 10 min, 2000 g for 20 min and 10,000 g for 30 min at 4 °C. Then supernatant was filtered through 0.22 μm filters (Millipore, MA, USA, SLGP033RB) to remove contaminating apoptotic bodies, microvesicles and cell debris. Clarified culture medium was then centrifuged in a Beckman Coulter Optima TM L-80XP Ultracentrifuge at 100,000 g at 4 °C for 70 min to pellet exosomes. The supernatant was carefully removed, and EVs-containing pellets were resuspended in1 mL of ice-cold PBS and pooled. A second round of ultracentrifugation [100,000 g at 4 °C for 70 min with a Type 50.2 Ti rotor (k-factor: 157.7)] was carried out, and the resulting EVs pellet resuspended in 100 μL of PBS and stored at −80 °C. The protein content of the concentrated EVs was determined using a BCA protein assay kit. Raw264.7-EVs were identified by transmission electron microscopy and NTA (NS3000, Worcestershire, UK), tetraspan molecules CD63 (ab10895, Abcam, Cambridge, UK), CD9 (ab92726, Abcam, Cambridge, UK), CD81 (18250-1-AP, Proteintech, Rosemont, USA) were verified by western blotting.