Fig. 2. LPS suppresses Notch1-IC transcriptional activity through NO in macrophage cells. (A-H) RAW264.7 (A,C,E,G) or alveolar macrophage (B,D,F,H) cells were transfected for 24 hours with the indicated combinations of expression vector for 4xCSL-Luc with Notch1-IC along with lacZ. (A-J) The cells were pre-treated with 100 µM L-NIL (A,B,E,F) or 2 mM L-NNA (C,D,G,H,I,J) for 30 minutes and then exposed to 5 µg/ml LPS for 24 hours. (A-D) NO released into the culture medium was then determined by the Griess method and represents nitrate+nitrite formation per 1x10⁶ cells. (E-H) 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. (I,J) The cell lysates were also subjected to immunoblotting analysis with anti-Hes1 or anti-Hes5 antibody. (K,L) RAW264.7 or alveolar macrophage cells were pre-treated with 2 mM L-NNA for 30 minutes and then exposed to 5 µg/ml LPS for 24 hours. (M) HEK293 cells were transfected with expression vector for Notch1-IC and RBP-Jk. The cells were then treated with 200 µM SNAP for 8 hours. (N) HEK293-neo or HEK293-bNOS cells were transfected with expression vector for Notch1-IC and RBP-Jk. The cells were pre-treated with 2 mM L-NNA for 30 minutes and then exposed to 20 mM L-Arg for 16 hours. The cells were crosslinked with formaldehyde and DNA was immunoprecipitated with the indicated antibodies. The immunoprecipitated DNA was analyzed by PCR using primers recognizing the Hes1 or Hes5 promoters. As a negative control, we also tested a sample with vehicle only and pre-immune IgG, and included an input sample.