Expression of EphB receptors and ephrin B ligands in primary human endothelial cells from the umbilical vein, aorta, and dermal microvasculature. (A) EphB1, EphB2, EphB3, EphB4, and EphB6 receptor and ephrin B1, ephrin B2, and ephrin B3 ligand mRNA detected by semiquantitative RT-PCR with specific primer pairs in HUVECs, HAECs, and HDMECs. Representative results from 3 experiments. (B) EphB1, EphB2, EphB3, EphB4, and EphB6 were detected by Western blotting with specific antibodies in cell lysates from murine NIH3T3 cells, human brain tissue (brain), HUVEC, human SK-N-MC neuroblastoma cell line (neuro), human A-204 rhabdomyosarcoma cell line (rhabd), and human SK-NEP-1 Wilms tumor cell line (wilms), HAEC, and HDMEC. Results shown, which are representative of 4 independent immunoblotting experiments, reflect reprobing of a single membrane after stripping; sample loading was evaluated by reprobing for actin. (C) Immunoblotting analysis of ephrin B2 and ephrin B3 expression in HUVECs, HAECs, HDMECs, and other samples—murine NIH3T3 cells, human brain tissue (brain), human SK-N-MC neuroblastoma cell line (neuro), human A-204 rhabdomyosarcoma cell line (rhabd), and human SK-NEP-1 Wilms tumor cell line (wilms). A single membrane was used for immune detection; sample loading was evaluated by probing with antiactin antibodies. Results are representative of 3 immunoblotting experiments.