Western Blot

Western blot analysis was implemented as previously described (Meng et al., 2015). Total protein was isolated from cultured HUVECs using the RIPA lysis buffer (Solarbio) and was quantified using BCA Protein Assay Kit (Thermo Fisher Scientific). Proteins (30 µg per lane) were resolved onto a 10% SDS–polyacrylamide gel and then transferred onto Clear Blot membrane-p (ATTO, Tokyo, Japan). The membranes were incubated with primary antibodies and horseradish peroxidase (HRP)–linked IgG secondary antibody (ab6721 or ab6728, Abcam, Cambridge, United Kingdom; dilution 1:10,000). The bound antibodies were detected by the enhanced chemiluminescence (ECL, Thermo Fisher Scientific) and analyzed using an image analyzer (LAS-4000, Fuji Photo Film, Minamiashigara, Japan). Primary antibodies against vascular endothelial growth factor (anti-VEGF, #MA1-16629; dilution 1:1,000), B-cell lymphoma 2 (anti-Bcl-2, #MA5-11757; dilution 1:50), Bcl-2–associated X protein (anti-Bax, #MA5-13994; dilution 1:100), and glyceraldehyde-3-phosphate dehydrogenase (anti-GAPDH, #AM4300; dilution 1:100) were purchased from Invitrogen (Toyko, Japan). Primary antibodies, including anti-Ki67 (#sc-23900; dilution 1:500), anti-poly (ADP-ribose) polymerases (anti-PARP, #sc-8007; dilution 1:1,000), and anti-Cleaved PARP (#sc-56196; dilution 1:1,000), were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, United States). The FBXW7 antibody (anti-FBXW7, #ab192328; dilution 1:1,000) was obtained from Abcam.