Rabbit anti tsg101

Cell or exosome lysates were prepared in 3-(N-morpholino)propanesulfonic acid (MOPS) buffer on ice for 30 min. Then, debris was removed by centrifugation for 20 min at 4°C. Protein concentration was measured with the bicinchoninic acid (BCA) assay (Sigma, St. Louis, MO, USA). After being electrophoresed in 10% Bis-Tris gel, proteins were transferred to the polyvinylidene fluoride (PVDF) membrane. The samples were then blocked in the nonspecific binding sites overnight. Following transfer to nitrocellulose membranes, samples were incubated with the primary antibodies as follows: rabbit anti-CK8 (1:200), rabbit anti-vimentin (1:200), rabbit anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (1:1,000), mouse monoclonal anti-CD63 (1:200), rabbit anti-Tsg101 (1:200), rabbit anti-calnexin (1:200), rabbit anti-Wnt11 (1:500), and rabbit anti-β catenin (1:500) (Abcam, Cambridge, MA, USA), Antibody labeling was identified using horseradish peroxidase (HRP)-conjugated secondary antibodies (Cell Signaling Technology, Boston, MA, USA). Images were analyzed by densitometry from Scion Image.

Exosomes, cells, or tissues after indicated treatments were harvested and subjected to radioimmunoprecipitation assay (RIPA) lysis buffer (Beyotime Biotechnology, China) supplemented with protease inhibitor cocktail (Roche). Purified protein was separated in 12% SDS-PAGE (120 V for stacking gel and 160 V for separation gel) and then transferred to a nitrocellulose membrane with an ice bath. The nitrocellulose membrane was blocked with 5% bovine serum albumin for 1 h and then incubated overnight with primary antibodies at 4°C. Antibodies used were mouse anti-GM130, rabbit anti-CD9, rabbit anti-TSG101, rabbit anti-PGC1α, and rabbit anti-GAPDH (all from Abcam). The membrane was then incubated for 1 h with the corresponding secondary antibodies at room temperature and visualized using the enhanced chemiluminescence (ECL) Prime western blotting detection reagent (GE Healthcare, Buckinghamshire, UK).

Proteins were extracted using RIPA lysis buffer (cat. no. HY-K1001; MedChemExpress) from JEG-3 cells and MSCs-Ex. The concentration of the isolated protein was quantified via BCA Protein Assay kit (cat. no. 23225; Thermo Fisher Scientific, Inc.). Isolated proteins (5 µg) were mixed with 5X SDS-PAGE protein loading buffer (cat. no. 20315ES05; Shanghai Yeasen Biotechnology Co., Ltd.). Proteins (20 µg) were separated on 12% SDS-acrylamide gels, followed by transferring onto PVDF membranes, which were incubated with 5% non-fat milk for 1 h at room temperature, and with rabbit anti-HIF-1α (1:1,000; cat. no. ab179483; Abcam), rabbit anti-CD9 (1:1,000; cat. no. ab236630; Abcam), rabbit anti-CD81 (1:1,000; cat. no. ab79559; Abcam), rabbit anti-LAMP-2B (1:1,000; cat. no. ab18529; Abcam), rabbit anti-TSG101 (1:1,000; cat. no. ab125011; Abcam) and rabbit anti-GAPDH (1:10,000; cat. no. 10494-1-AP; ProteinTech Group, Inc.) overnight at 4°C. Subsequently, membranes were incubated with horseradish peroxidase-conjugated mouse anti-rabbit IgG (1:5,000; cat. no. BM2006; Boster Biological Technology) for 1 h at room temperature. Protein bands were determined via ECL western blot detection reagents (Thermo Fisher Scientific, Inc.). The protein gray value was calculated using ImageJ (Version 1.5.3; National Institutes of Health).