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Fig. 7. Mapping of nitrated tyrosine residues in Notch1-IC. (A) HEK293 cells were transfected with Flag–Notch1-IC, Flag–Notch1-IC-N, Flag–Notch-IC-NC or Flag–Notch1-IC-C, after which the cells were exposed to 200 µM SNAP for 8 hours. The cells were lysed and immunoprecipitated against anti-Flag antibody and immunoblotted against anti-nitrotyrosine antibodies. (B) RAW264.7 cells were transfected for 24 hours with the indicated expression vectors for Flag–Notch1-IC (Y1905/1928F), Flag–Notch1-IC (Y1928/2064F) or Flag–Notch1-IC (Y1905/2064F) and then exposed to 5 µg/ml LPS for 24 hours. The cells were lysed and immunoprecipitated against anti-Flag antibody. The immunocomplexes were analyzed via SDS-PAGE and immunoblotting against anti-nitrotyrosine or anti-Flag monoclonal antibody. (C) NIH3T3 and RAW264.7 cells were transfected with the indicated expression vector for 4xCSL-Luc, β-galactosidase and Notch1-IC (Y1905F), and then exposed to 200 µM SNAP for 8 hours or 5 µg/ml LPS for 24 hours, respectively. The cells were then lysed and assayed for luciferase activity. The activity of the luciferase reporter in each of the samples was 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. (D) HEK293 cells were transfected with Flag–Notch1-IC (Y1905F) and Myc–RBP-Jk, after which the cells were exposed to 200 µM SNAP for 8 hours. The cells were lysed and immunoprecipitated against anti-Flag or anti-Myc antibody and immunoblotting was conducted against anti-Myc or anti-Flag antibody.

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