Anti epha2

Cell extracts were prepared in IP150 lysis buffer (20 mM Tris-HCl pH 7.6, 150 mM NaCl, 0.5% Nonidet P-40, 10% Glycerol) containing protease inhibitors (1 mM Na₂VO₄, 10 mM NaF, 2 mM PMSF, 5 μg/ml Leupeptin, 10 μg/ml Aprotinin, 1 μg/ml Pepstatin A) (Roche, Switzerland). Equal amounts of protein were separated by SDS-PAGE and transferred onto PVDF membranes (PALL Life Sciences, USA). Membranes were subsequently incubated with the appropriate primary antibodies overnight at 4 °C, followed by incubation with peroxidase-conjugated secondary antibodies for 1 h at room temperature. Protein bands were visualized using the ECL chemiluminescent detection system (iNtRON Biotechnology, Korea). For immunoprecipitation of protein complexes, cell extracts were precleared with protein G-Sepharose beads (GE Healthcare) and incubated with the appropriate antibodies. Immune complexes were then analyzed by immunoblotting, which was performed using the following antibodies: anti-Ephexin1 and anti-β-actin from Abcam (Cambridge, MA, USA); anti-ERK1/2, antiphospho-ERK1/2 (T202/Y204), anti-Ki67, anti-EGFR, and antiphospho-EphA2 (S897) from Cell Signaling (Danvers, MA, USA); anti-HA, antimyc, anti-V5, and anti-EphA2 from Santa Cruz (Dallas, TX, USA); anti-FLAG (M2) from Sigma-Aldrich; and anti-Ras from BD Biosciences (San Jose, CA, USA).

The expression of cell surface molecules was determined by flow cytometry (Fortessa; Becton Dickinson) using a fluorescein isothiocyanate (FITC)- or phycoerythrin (PE)-conjugated MAb specific for HLA-DR, CD1a, CD11b, CD14, CD91, CD80, CD86, CD83, CD209 (DC-SIGN), or CD207 (langerin) or polyclonal anti-EphA2 (Santa Cruz Biotechnology) for 30 min at 4°C, and the cells were then fixed with 1% paraformaldehyde. Cells stained with isotype immunoglobulin served as controls. Optimal photomultiplying tube (PMT) voltage settings for each fluorescence channel were determined on the day of the experiment by using cytometer setup and tracking (CST) beads (Standardization and Tracking beads; BD Biosciences). These voltage values are set to give the lowest coefficient of variation (CV) for that individual fluorescence channel.

Protein extracts were obtained using a protein extraction kit. Protein concentration was determined by the Bradford assay kit (Bio-Rad, Hercules, CA). The protein extract was electrophoresed on a sodium dodecyl sulphate - polyacrylamide (SDS - PAGE) gel and transferred to polyvinylidene fluoride (PVDF) membranes. After incubation with 5% dry non-fat skimmed milk to block non-specific binding, the membranes were exposed to specific anti-HIF-1α (1∶500), anti-HIF-2α (1∶1000), anti-VEGFA (1∶200), anti-EphA2 (1∶100), and anti-β-actin (1∶1000, Santa Cruz Biotechnology) monoclonal antibodies overnight at 4°C. The membranes were washed and exposed to peroxidase-conjugated anti-IgG secondary antibody (1∶5000). Finally, the immune complexes were developed using an enhanced chemiluminescence detection kit according to the manufacturers instructions (Pierce, Waltham, MA) and the GelGDoc2000 imaging system (Bio-Rad Germany) was employed to analyze the bands, and the protein levels by the relative optical density.