Rabbit anti tsg101

The proteins from UFs‘ EVs and the endometrium were extracted by RIPA buffer (Beyotime, Cat. No. P0013B) supplemented with 1% PMSF (Beyotime, Cat. No. ST506). The sample was denatured by heating, separated by SDS-PAGE, transferred to a polyvinylidene difluoride (PVDF) membrane (Millipore, Cat. No. IPVH08110), washed four times (5 min each time) with TBST, and blocked with 5% skim milk in TBST for 2 h at room temperature. The following antibodies were incubated with the membranes after being washed four times with TBST: rabbit anti TSG101 (ZEN-BIOSCIENCE, Cat. No. abs127362, 1:1000 in TBST), rabbit anti HSP70 (Proteintech, Cat. No. 25682-1-AP, 1:1000 in TBST), and rabbit anti Calnexin (Proteintech, Cat. No. 10427-2-AP, 1:1000 in TBST) overnight at 4 °C. After being washed four times with TBST, the membranes were incubated with HRP-labeled Goat Anti-Rabbit secondary antibodies (Beyotime, Cat. No. A0208, 1:1000 in TBST) for 2 h at room temperature. The images of the membranes were captured by a UVP system (Upland) after they were treated with an enhanced chemiluminescence (ECL, Beyotime, Cat. No. P0018S) reagent.

Radioimmunoprecipitation assay (RIPA) buffer containing protease and phosphatase inhibitors (BestBio Cat No. BB-3101) was used to extract proteins. The protein concentration was assessed using the Rapid Gold BCA Protein Assay Kit (Thermo Fisher). Western blotting analysis was performed by loading 15 µg of lysate onto sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels, transferring the gels to polyvinylidene difluoride (PVDF) membranes (Millipore), and incubated with rabbit anti-GDF5 (1:1000, #A13167; ABclonal), rabbit anti-PPARγ (1:1000, #2443; CST), rabbit anti-FASN (1:1000, #D262701; Sangon Biotech), rabbit anti-C/EBPα (1:1000, #2295; CST), rabbit anti-FABP4 (1:1000, #2120; CST), rabbit anti-CD36 (1:1000, #ab1336-25; Abcam), rabbit anti-GAPDH (1:5000, #BS65529; Bioworld), rabbit anti-CD9 (1:1000, #AP68-965-100; Abcepta), rabbit anti-CD63 (1:2000, #D160973; Sangon Biotech), rabbit anti-TSG101 (1:2000, #381538; ZEN BIO), rabbit anti-Alix (1:1000, #D262028; Sangon Biotech) or rabbit anti-Calnexin (1:1000, #D262986; Sangon Biotech). Afterward, goat anti-rabbit secondary antibody (1:50000, # BS13278, Bioworld) conjugated with HRP was used. GAPDH levels served as the loading control. The amount of protein was measured using ImageJ software.