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Fig. 5. NO prevents the physical interaction between Notch1-IC and RBP-Jk both in vitro and in vivo. (A,B) HEK293 cells were transfected with Myc–Notch1-IC and Flag–RBP-Jk, and then the cells were exposed to 200 µM SNAP for 8 hours. After 48 hours, the cells were lysed and immunoprecipitated against anti-Myc (A) or anti-Flag (B) monoclonal antibody. The immunocomplexes were analyzed via SDS-PAGE and immunoblotting against anti-Flag monoclonal antibody. (C,D) HEK293 cells were transfected with Myc–Notch1-IC and Flag-RBP-Jk. The cells were lysed and immunoprecipitated against anti-Flag (C) or anti-Myc (D) monoclonal antibody, and then exposed to 200 µM SNAP for 1 hour on ice. The immunocomplexes were analyzed via SDS-PAGE and immunoblotting against anti-Myc monoclonal antibody. (A-D) The expression of Notch1-IC or RBP-Jk was analyzed via immunoblotting using anti-Myc or anti-Flag monoclonal antibody, respectively. (E) Recombinant GST and GST–Notch1-IC proteins, immobilized on glutathione-agarose beads, were exposed to 200 µM SNAP for 1 hour on ice, and extensively washed to remove remnant SNAP. HEK293 cells were transfected with RBP-Jk, and then the cells were lysed and added to the immobilized proteins. The beads were extensively washed, eluted and analyzed using SDS-PAGE immunoblotting against anti-Flag monoclonal antibody. Coomassie blue staining represents immobilized proteins. (F) RAW264.7 cells were pre-treated with 2 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. The immunocomplexes were analyzed via SDS-PAGE and immunoblotting against anti-RBP-Jk antibody. (G) Primary alveolar macrophage cells from 5 µg/ml LPS-injected rats were lysed and immunoprecipitated against anti-Notch1-IC antibody. The immunocomplexes were analyzed via SDS-PAGE and immunoblotting against anti-RBP-Jk antibody. (F,G) 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. (H) HEK293 cells were transfected for 40 hours with the indicated combinations of expression vector for Myc–Notch1-IC, Flag–RBP-Jk and HA-Mastermind, and then the cells were exposed to 200 µM SNAP for 8 hours. After 48 hours, the cells were lysed and immunoprecipitated against anti-Myc monoclonal antibody. The immunocomplexes were analyzed via SDS-PAGE and immunoblotting against anti-Flag or anti-HA monoclonal antibody. The expression of Notch1-IC, RBP-Jk or Mastermind (Mam) was analyzed via immunoblotting using anti-Myc, anti-Flag or anti-HA monoclonal antibody, respectively.
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