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Fig. 6. Nitration of the Notch tyrosine residue 1905 mediated a conformational change. (A) HEK293 cells were transfected with Myc–Notch1-IC, after which the cells were exposed to 200 µM SNAP for 8 hours. The cells were lysed and immunoprecipitated against anti-Myc monoclonal antibody. The immunocomplexes were analyzed via SDS-PAGE and immunoblotting against anti-nitrocysteine or anti-nitrotyrosine antibodies. (B) Various concentrations of purified Notch1-IC (0, 50, 75, 100, 125, 150 or 200 µM) were pre-treated with 200 µM SNAP for 30 minutes on ice and then incubated with 0.20 mM DTNB. Maximum absorbance (Abs) was obtained at 412 nm. (C) The purified Notch1-IC proteins were exposed to 200 µM SNAP and dissolved in 50 mM Tris-HCl at a pH of 7.4. The intrinsic fluorescence spectra were acquired at an excitation wavelength of 278 nm, and excitation and emission slits of 5 nm. We conducted an emission wavelength scan from 300 nm to 400 nm. (D) RAW264.7 cells were pre-treated with 1 mM L-NNA for 30 minutes and then exposed to 5 µg/ml LPS for 24 hours. The cells were lysed and immunoprecipitated against anti-Notch1-IC antibody. (E) Primary alveolar macrophage cells from 1 mM L-NNA- and 5 µg/ml LPS-injected rats were lysed and immunoprecipitated against anti-Notch1-IC antibody. (D,E) The immunocomplexes were analyzed via SDS-PAGE and immunoblotting against anti-nitrotyrosine antibody. The expression of Notch1-IC or RBP-Jk was analyzed via immunoblotting using anti-Notch1-IC or anti-RBP-Jk antibody, respectively. Probing with an antibody to β-actin was used as a loading control.
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