Image lab system version 5

After 1 week induction, cells were rinsed with pre-cooled PBS and lysed using radioimmunoprecipitation assay lysis buffer (RIPA, Boster, China) containing 1% phosphatase and protease inhibitors. The protein concentration of the lysate was determined using a BCA protein assay kit. Cell lysates (20 μg protein) were resolved onto SDS-polyacrylamide gels (8–10%) and transferred onto 0.45-μm polyvinylidene difluoride (PVDF) membranes (Millipore, USA). After blocked with 5% bovine serum albumin (BSA) in Tris-buffered Saline-Tween solution (TBST) for 1 h at room temperature, the blots were incubated with the specific primary antibodies (OPN, RUNX2, COLI, VEGFR2, vWF, CD31, WNT1, and LRP-6 at 1:1000 dilution, β-catenin at 1:2000 dilution, Abcam, UK; GAPDH at 1:500 dilution, Boster, China) at 4 °C overnight. The membranes were next incubated with the corresponding horseradish peroxidase-conjugated goat anti-rabbit (1:5000 dilution) or goat anti-mouse (1:5000 dilution) antibodies (Boster, China) at 37 °C for 1 h after rinsing with TBST. The blots were then performed with an enhanced chemiluminescence (ECL, Thermo Fisher Scientific, USA). Relative expression was quantified using the Image Lab system version 5.1 (Bio-Rad Laboratories, USA) and normalized to GAPDH.