Figure 2
The Rap signal is involved in Notch S2 processing by sustaining Adam10 maturation via proprotein convertase activity.
(a) FL0/rtTA-Sipa1 cells were cultured in the absence (fine line) or presence (solid line) of Dox (1 μg/mL) and the surface expression of Notch1 was analyzed with flow cytometry. These cells were cultured with or without varying dosed of Dox for 3 days and the lysates were immunoprecipitated with anti-Notch1 antibody followed by immunoblotting with S2-specific (V1711) antibody. The mean signal intensities of Notch S2 in 3 independent experiments are shown. *; p < 0.01. (b) FL0/rtTA-Sipa1 cells were cultured in the absence or presence of Dox for 3 days and the lysates were immunoblotted with anti-Adam10 antibody. Adam10 transcripts were assessed with qRT-PCR in the aliquots of cells. The mean signal intensities of mature (m)-Adam10 and the relative transcripts in 3 independent experiments are shown. *; p < 0.01. NS; not significant. (c) The cells in (b) were immunoblotted and FACS analyzed with anti-Dll1 antibody. (d) FL0/rtTA-Sipa1 cells were cultured in the absence or presence of Dox for 3 days and proprotein convertase activity of the cell lysates was assessed with the use of a fluorescence-labeled synthetic substrate in the presence or absence of EDTA. In (a), (b) and (c), relevant parts of immunoblot images were cropped from full-length blots shown in Figure S8.
