The temporal and dose effects of ephrinB2 on expressions of syntenin and syndecan-1 in HUAECs by RT-PCR and Western blot. (A-D) To confirm the results of differentially expressed genes obtained by the cDNA microarray technique, each candidate gene was further analyzed with RT-PCR to test dose dependence and time dependence. Syntenin and syndecan-1 were the only 2 genes up-regulated in dose- and time-dependent manners. Neutralizing antibody (10 μg/mL) against the N-terminal of EphB4 receptor suppressed the up-regulation of syntenin and syndecan-1 by ephrinB2 (B,D). G3PDH was used as an internal control. The sizes of the PCR products were 388, 416, and 983 bp for syntenin, syndecan-1, and G3PDH, respectively. (E) Western blot of syntenin and syndecan-1 in HUAECs treated with different doses of ephrinB2. HUAECs were treated with preclustered ephrinB2 (0 to 500 ng/mL) for 24 hours. The cell lysates were then collected and analyzed with Western blot for syntenin and syndecan-1. The molecular weight for syndecan-1 is about 110 kDa for the major band and about 90 kDa for the minor band. The detected band for syntenin is about 33 kDa. Glypican-1 (another cell membrane proteoglycan on endothelial cells) and β-actin were used as controls.