Western Blot Analysis of Protein Markers

Twenty to 80 μg of protein lysate was separated electrophoretically on denaturing SDS-polyacrylamide gel, transferred to nitrocellulose membranes, and probed with primary antibodies. Blots were exposed to horseradish peroxidase-conjugated secondary antibodies and visualized by an ECL detection system according to the manufacturer’s protocol (Amersham Biosciences, Piscataway, NJ). For loading control, the initial western blot was placed in Restore Western stripping buffer (Thermo Scientific) for 15 min to remove the antibody (primary and secondary), washed in water for 5 min, blocked with 5% milk for 1 hour, and probed with β-actin antibody (Santa Cruz Biotechnology, Santa Cruz, CA; Catalog # sc-130301) [20 (link),24 (link)]. The primary antibodies used in western blot included NRP2, GAPDH (Santa Cruz Biotechnology, Santa Cruz, CA. NPR2 catalog # sc-13117; GAPDH catalog # sc-137179); VEGF, DKK3 (Abcam, Cambridge, MA. VEGF catlog # ab46154; DKK3 catalog # ab136101); Wif-1 (Cell Signaling Technology, Danvers, MA, Catalog # 5652); Each western blot was repeated 3 times. Blots were quantitated by densitometry using Image J (Software, NIH, Bethesda, MD, USA) and normalized to a housekeeper marker β-actin or GAPDH [36 (link)]. The intensity of each tested marker is presented as a ratio of a tested mark/β-actin or GAPDH.