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Fig. 3. NO modulates Notch transcriptional activity. (A,B) NIH3T3 cells were transfected for 40 hours with the indicated combinations of expression vector for 4xCSL-Luc, Hes1-Luc or Hes5-Luc and Notch1-IC along with lacZ. The cells were then treated with 200 µM SNAP (A) or 200 µM NAP (B) for 8 hours. The cell lysates were subjected to immunoblotting analysis with the anti-Hes1 or anti-Hes5 antibody. (C,D) HEK293-neo, -eNOS or -bNOS cells were transfected for 32 hours with 4xCSL-Luc and Notch1-IC along with lacZ. The cells were pre-treated with 2 mM L-NNA for 1 hour and then exposed to 20 mM L-Arg for 16 hours. (C) NO released into the culture medium was determined by the Griess method and represents nitrate+nitrite formation per 1x10⁶ cells. (D) Cells were lysed and assayed for luciferase activity. The activity of the luciferase reporter in each of the samples was then normalized according to the β-galactosidase activity measured in the same sample. These results represent the means ± average deviation of triplicates from one of three independent experiments. (E) HEK293-neo, HEK293-eNOS and HEK293-bNOS cells were transfected with expression vector for EN1 and then exposed to 20 mM L-Arg for 16 hours. The cell lysates were subjected to immunoblotting analysis with the anti-V1744 antibody.

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