Fig. 4

Notch1^C1133Y mutation causes reduced S1-cleavage of the Notch1 receptor and accumulation of receptor protein in the endoplasmic reticulum. A Notch1 protein expression pattern was evaluated by western blot using a Notch1 primary antibody which could detect endogenous Notch1 protein and recognize both full-length Notch1 (FL, 300 kD, S1-uncleaved premature form) and its cleaved transmembrane/intracellular region (NTM, 120 kD, S1-cleaved and S2/S3-cleaved form). B The subcellular location of Notch1 receptors in HN6 cells was assessed by immunofluorescence. The Notch1-FITC staining revealed that Notch1 protein in Notch1^WT-transfected cells was localized in the cytoplasm as well as on the cell surface (a, d), while Notch1 protein in C1133Y-mutated cells was only localized in the cytoplasm (a′, d′). Costaining of Notch1 with the Golgi-marker GM130 (b, b′) demonstrated overlapped staining in Notch1^WT-transfected cells (c), but did not showed overlap in Notch1^C1133Y-transfected cells (c′). Costaining of Notch1 with ER-marker Calnexin (e, e′) showed strong overlapped staining in both Notch1^WT and Notch1^C1133Y-transfected cells (f, f′). Scale bars are 10 μm. C The localization of Notch1 in cytoplasm or on cell surface was assessed in 100 cells, and the percent of cells was shown. D Overlapped staining of Notch1 with Golgi-marker GM130, or Notch1 with ER-marker Calnexin was counted. Percentages of localization were calculated from three independent experiments