Cd63 25682 1 ap

Exosomes were isolated by ultracentrifugation. Brie y, the conditioned supernatants of MSCs pretreated with or without TSA were collected and centrifuged at 300 ×g for 10 min. The supernatant was centrifuged at 10000 ×g for 30 min and ltered through a sterile 0.22-μm pore lter. The ltered solution was ultracentrifuged at 120,000 ×g for 2 h. Lastly, the supernatant was discarded to obtain the exosome precipitate [25, 26] . The structure of exosomes was observed by transmission electron microscopy (TEM) analysis using a Hitachi HT7700 (Hitachi, Japan) and their size distribution was determined by dynamic light scattering (DLS) using a DynaPro NanoStar (WYATT, USA). The exosomes were identi ed using western blotting with the marker proteins CD9 (20597-1-AP; Proteintech, Chicago, USA), CD63 (25682-1-AP; Proteintech), and Alix (12422-1-AP; Proteintech). The protein concentration in exosomes was determined using the bicinchoninic acid kit (Thermo Fisher Scienti c, Waltham, MA, USA).

5× loading buffer was added to the solution in a 4:1 ratio and denatured at 100°C for 5 min. SDS-PAGE was electrophoresed at 75 V for 130 min and then transferred to the membrane at 300 mA for 1 h. After 5 min of TBST rinsing, 5% skimmed milk was blocked for 1.5 hours, TBST was rinsed for 15 min, and CD63(25682-1-AP, 1:300, proteintech, USA), CD81 (66866-1-Ig, 1:3000, proteintech, USA), CD9 (20597-1-AP, 1:300, proteintech, USA), GAPDH(60004-1-Ig, 1:5000, proteintech, USA), PI3K (67071-1-Ig, 1:5000, proteintech, USA), p-Akt(66444-1-Ig, 1:5000, proteintech, USA), Ang-1 (ab183701, 1:10000, abcam, UK), Tie-2(19157-1-AP, 1:500, proteintech, USA), and b-actin (60008-1-Ig, 1:5000, proteintech, USA) primary antibodies were added and incubated overnight at 4°C. The PVDF membrane was taken out and rinsed with TBST. The secondary antibody HRP goat anti-mouse IgG (SA00001-1, 1:5000, Proteintech, USA) or HRP goat anti-rabbit IgG (SA00001-2, 1:6000, Proteintech, USA) was added, incubated at 25°C for 1.5 h, and rinsed with TBST. The Western strip image was scanned and analysed by a chemiluminescence imaging system (Guangzhou Qinxiang Company). GAPDH or b-actin was used as an internal control.