Western Blotting Analysis of Angiogenic Markers

Western blotting experiments were performed according to previous studies [40 (link)]. Briefly, proteins (50 μg) were loaded into a 10% SDS-PAGE gel and electrotransferred to a polyvinylidene difluoride membrane (Millipore, USA). Membranes were incubated overnight at 4°C in primary antibodies recognizing Jagged-1 (1 : 1000, Cell Signaling Technology), Notch-1 (1 : 1000, Cell Signaling Technology), Hes-1 (1 : 1000, Cell Signaling Technology), CD31 (1 : 1000 dilution, Abcam), zonula occludens-1 (ZO-1) (1 : 1000 dilution, Abcam), and vWF (1 : 500, Santa Cruz Biotechnology). Membranes were washed in TBS and incubated in secondary antibodies conjugated to horseradish peroxidase (HRP) (1 : 1000, Cell Signaling Technology) for 2 h at room temperature. GAPDH (1 : 1000 dilution, Abcam) was used as an endogenous loading control. The ImageQuant LAS 4000 mini detection system (GE Healthcare, Buckinghamshire, UK) was used for quantified densitometric analysis, and ImageJ software (National Institutes of Health, Bethesda, MD) was used to analyze results.