Figure 4.

Potentiation of Notch-target gene expression in the absence of FADD. (a) FACS was performed to analyze expression of CD25 expression on the cell surface of DP, CD4 SP and CD8 SP cells. Shown are representative data from three independent experiments. (b) Notch1 western blot (left, α-tubulin expression served as a loading control (bottom panel)) and quantitative PCR for Notch1 and its target genes expression of thymocytes from Lck-FADD mice with littermate control (right, n>3, ***P<0.001). (c) DN3, DN4 and DP thymocytes from Lck-FADD mice and littermate control were isolated by FACS sorting. mRNA was generated and examined by quantitative PCR for Notch1, CD25, Hes1 and Deltex1 mRNA expression levels, and normalized relative to the sorted wild-type population with the lowest expression of each gene (=1). The data shown are from three Lck-FADD mice and littermate control, each from independent sorts and independent Q-PCR. Error bars reflect S.E.M. (d) DN3, DN4 and DP thymocytes from Lck-FADD mice and littermate control were isolated by FACS sorting and stained for FADD and DAPI for immunofluorescence analysis of FADD and Notch1 expression. Pictures were taken on a microscope at × 1200 magnification. (e) Immunoblot analysis of Notch1 in WT and FADD^−/− MEFs after exogenous FADD was introduced for 24 h. The data shown are from three independent experiments