HLX up-regulates genes for repellent cell-guidance molecules and a transcriptional repressor of the Notch signaling pathway. (A) Real-time RT-PCR analysis of UNC5B, PLXNA1, SEMA3G, and HES1 mRNA: HUVECs were transduced for 4, 8, 16, and 32 hours with recombinant adenoviruses encoding HLX (Ad.HLX) or control viruses (Ad.con) using an MOI of 20. Total RNA was isolated and real-time RT-PCR performed as described in “Real-time RT-PCR analysis.” Values were normalized to β2-microglobulin mRNA as internal standard. Mean values ± SD calculated from triplicate wells are depicted as fold induction of the mRNA levels obtained after infection with Ad.HLX (■) or Ad.con (○) compared with noninfected cells. Results of 1 representative experiment of 3 independently performed are shown. (B) Western blot analysis of HLX: Total cell lysates were prepared from HUVECs nontransduced (0 hours) or tranduced with Ad.HLX (4, 8, 16, and 32 hours) or with Ad.con for 32 hours using an MOI of 20. Cells were harvested and proteins were separated by SDS- PAGE. Membranes were probed with anti-HLX and anti-GAPDH antibodies.