Cd81 ab109201

Tendon-bone junction tissues were cut into pieces, and proteins were harvested from the tissues using ice-cold RIPA lysis. Then, the protein concentration was measured using a BCA Protein Assay kit (Beyotime, China). The protein samples were separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto polyvinylidene difluoride (PVDF) membranes, which were blocked with 5% bovine serum albumin (BSA; Sangon Biotech, China) for 1 h. Later, primary antibody was added to incubate the membranes at 4°C overnight. Primary antibodies CD206 (ab64693, 1: 1000), CD81 (ab109201, 1: 500), CD9 (ab92726, 1: 800), and tumor susceptibility gene 101 (TSG101; ab125011, 1: 1000) were purchased from Abcam, and Arg1 (93668S, 1: 1000) was purchased from Cell Signaling Technology. Then, the membranes were incubated with HRP-conjugated secondary antibody (Beyotime, A0208, 1: 1000) at 37°C for 2 h. Finally, the reaction solution was added for exposure analysis.

Protein content from all samples was quantified using a bicinchoninic acid protein assay kit (23225; Thermo Fisher). Western blot analysis was performed using standard procedures, was detected using an enhanced chemiluminescence western blotting detection kit (32106; Thermo Fisher), and was quantified by scanning densitometry. Antibodies against phosphorylated (p)-protein kinase B (Akt) (Ser473) (#4060), Akt (#4685), p-extracellular signal-regulated kinase (ERK) (Thr202/Tyr204) (#4370), ERK (#4695), VEGF receptor-2 (VEGFR2, #9698), p-VEGFR2 (Tyr1175) (#3770), β-actin (#4970), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH, #5174) were purchased from Cell Signaling Technology (Danvers, MA, USA). CD36 (18836), CPT1A (15184), carnitine palmitoyl-transferase 1B (CPT1B; 22170), VEGF (19003), apolipoprotein A1 (APOA1; 14427), and golgi matrix protein 130 (GM130; 11308) were purchased from Proteintech. AGPAT1 (GTX55496) was purchased from GeneTex. CD31 (ab213175), tumor susceptibility 101 (TSG101, ab125011), and CD81 (ab109201) were purchased from Abcam. GAPDH or β-actin was used as a loading control.