Anti ephrin a3

Proteins (50 μg) were extracted from FFPE tissues and separated as mentioned. Primary antibodies were incubated overnight at 4°C as follows: anti-Ephrin-A3 (1:50 dilution; Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-PTP1B (1:200 dilution; Abcam, Cambridge, UK), and anti-VEGF (1:200 dilution; Abcam, Cambridge, UK). Secondary antibodies were used as follows: anti-rabbit for Ephrin-A3, PTP1B, and VEGF (1:2,000 dilution, Cell Signaling Technology, Danvers, MA, USA). Beta-actin was used as a loading control.

Tumor tissues were fixed in 3.7% paraformaldehyde for 24 h and embedded with paraffin, prepared as 4-μm sections, and mounted on slides. Antigens were retrieved by boiling in citrate buffer (DakoCytomation, Glostrup, Denmark) for 5 min. Primary antibodies were incubated overnight at 4°C as follows: anti-luciferase (1:500 dilution; Abcam, Cambridge, UK), anti-HIF-1α (1:100 dilution; Novus Biologicals, Littleton, CO, USA), and anti-Ephrin-A3 (1:500 dilution; Santa Cruz Biotechnology, Santa Cruz, CA, USA). Secondary antibodies were used as follows: biotinylated anti-goat for luciferase (1:500 dilution; Dako, Glostrup, Denmark) and biotinylated anti-mouse for HIF-1α and Ephrin-A3 (1:500 dilution; Dako, Glostrup, Denmark). An avidin–biotin peroxidase complex was used to amplify the signal, followed by development using DAB and counterstaining with hematoxylin.