Rabbit anti human endostatin antibody

All HREC groups were collected 72 h after transfer. Briefly, the proteins were separated via sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) in 12% (w/v) polyacrylamide gels and electrotransferred to Millipore polyvinylidene difluoride membranes (Millipore, Bedford, MA). The completion of the protein transfer from the gels to the membranes was verified by staining the gels with Coomassie Blue R-250. The membranes were blocked in Tris-buffered saline containing 0.05% Tween-20 and 5% nonfat dry milk for 60 min at room temperature. Then, the blots were incubated with a 1:1,000 dilution of a rabbit anti-human endostatin antibody (Abcam, London, UK), rabbit anti-human VEGF antibody (Abcam), or rabbit anti-human ES antibody (Abcam) for 1.5 h at room temperature in blocking solution. The membranes were washed in Tris-buffered saline and incubated with horseradish peroxidase-conjugated goat anti-rabbit immunoglobulin G (IgG; 1:3,000) in blocking solution for 1.5 h. The blots were examined using a chemiluminescent substrate (Santa Cruz Biotechnology, Santa Cruz, CA) according to the manufacturer’s instructions. The protein bands were normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and all of the blots were quantified using Quantity One software (Bio-Rad, Hercules, CA).

Both the control and treated HUVECs were collected 48 h after being transferred. Briefly, the proteins were separated by SDS-PAGE on 12% (w/v) polyacrylamide gels and electrotransferred onto Millipore polyvinylidene difluoride membranes (Millipore, Bedford, MA, USA). The completion of protein transfer from the gels to the membranes was verified by staining the gels with Coomassie Blue R-250. The membranes were blocked in Tris-buffered saline containing 0.05% Tween 20 and 5% nonfat dry milk for 60 min at room temperature. The blots were then incubated with 1:1000 rabbit-anti-human endostatin antibody (Abcam, Cambridge, UK), rabbit-anti-human VEGF antibody (Abcam, Cambridge, UK) or rabbit-anti-human caspase-3 antibody (Abcam, Cambridge, UK) for 1.5 h at room temperature in blocking solution. Then, the membranes were washed in Tris-buffered saline and incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG (1:3000) in blocking solution for 1.5 h. The blots were examined using a chemiluminescent substrate (Santa Cruz Biotechnology, Santa Cruz, CA, USA) according to the manufacturer's instructions. The protein bands were normalized to GAPDH, and all blots were quantified using the software Quantity One (Bio-Rad, Hercules, CA, USA).