Anti cd47

To examine the expression of the proteins in both cell lines, SKOV3-CDDP and SKOV3 cells were seeded at 2 × 10⁵ cells per well in 6-well plates and cultured overnight. The two cell lines were treated with PBS, miR497, miR497-HENPs, TP, TP-HENPs and miR497/TP-HENPs for 48 h. Then, 150 µl RIPA buffer (Sigma–Aldrich) was added and lysed on ice for 5 min. The lysate was collected and centrifuged at 12,000 g for 15 min at 4 °C. The protein content in the supernatant was quantified by a BCA protein assay kit. Protein samples were separated by SDS–PAGE and transferred to PVDF membranes, and the membranes were blocked with 5% blocking buffer for 1 h and then incubated overnight at 4 °C with the following primary antibodies: anti-calnexin, anti-TSG101, anti-CD9, anti-CD47, anti-GAPDH, anti-PI3K, anti-p-PI3K and anti-mTOR (purchased from Abcam) and anti-p-mTOR, anti-AKT, anti-p-AKT and HIF-α (obtained from CST). The membranes were washed three times, and then at room temperature, the secondary antibody was incubated for 1 h. All strips were visualized using a Bio–Rad Imaging System (Bio–Rad, USA).

Liver tissues were homogenized in RIPA buffer (Sigma, St. Louis, MO, USA) plus protease and phosphatase inhibitors (Pierce, Waltham, MA, USA). After concentrations were measured using a BCA Assay (Pierce, Waltham, MA, USA), 30 μg protein/well was subjected to SDS-PAGE gel under reducing conditions and transferred onto a nitrocellulose membrane. After blocking, the membrane was incubated with anti-β-actin (1:5000 dilution; Santa Cruz, Dallas, TX, USA) and anti-CD47 (1:1000 dilution; Abcam, Cambridge, MA, USA) at 4 °C overnight. After washing, the membrane was incubated with horseradish peroxidase-conjugated secondary antibodies (Jackson Labs, Bar Harbor, ME, USA). The reaction was visualized by using an enhanced chemiluminescence system (Pierce, Waltham, MA, USA).