Western-blot analysis was performed as previously described45 (link). Briefly, ECFC were grown on plates treated or not with 50 ng/mL sCD146, then washed in PBS, scraped off the plates and lysed with 200 μl denaturing lysis buffer (150 mM NaCl, 10 mM TrisHCl pH 8, 1 mM EDTA, NP40 10% and protease inhibitors) for 10 minutes at 4 °C. After centrifugation (12,000 g, 10 minutes, 4 °C) to eliminate cell debris and nuclei, proteins were quantified by protein assay (BCA Protein Assay Kit, Pierce). Then, 30 μg of protein were mixed with NuPAGE lithium dodecylsulfate (LDS) sample buffer (Invitrogen) and NuPAGE sample-reducing agent (Invitrogen). The samples were separated by electrophoresis on 4–12% gradient NuPage SDS-polyacrylamide gels(Life Technologies). After transfer, membranes were blocked with BSA 4% and incubated with a primary antibody and a secondary antibody coupled to peroxidase before detection with the ECL kit (Pierce).
Antibodies against p16INK4a, p21WAF, SIRT1, p53, VE-Cadherin, α-SMA, Vimentin and Actin were purchased from Cell Signaling Technology. Antibodies against PECAM-1, Oct-4, Sox-2, KLF-4 and c-MYC were purchased from Santa Cruz Biotechnology. The antibody against Nanog was purchased from R&D systems. All antibodies were used at the recommended dilution for immunoblotting.
Antibodies against p16INK4a, p21WAF, SIRT1, p53, VE-Cadherin, α-SMA, Vimentin and Actin were purchased from Cell Signaling Technology. Antibodies against PECAM-1, Oct-4, Sox-2, KLF-4 and c-MYC were purchased from Santa Cruz Biotechnology. The antibody against Nanog was purchased from R&D systems. All antibodies were used at the recommended dilution for immunoblotting.