Figure 5.

The increase of NOTCH expression coupled with decrease of NKAP. (a) DN3 and DN4 thymocytes from three wild-type and Lck-FADD mice were isolated by FACS sorting. mRNA was generated and examined by quantitative PCR for E2-2 and Id3 mRNA expression levels, and normalized relative to the sorted wild-type population with the lowest expression of each gene (=1). (b–e) Cryosections of thymi were stained with Abs and analyzed by immunofluorescence microscopy to detect the expression of FADD, Notch1 and Notch ligands in the thymus of Lck-FADD mice and littermate control. Scale bars: (b and e) 20 μm; (d) 50 μm; (c) 100 μm. (f) NKAP western blots and immunofluorescence analyze were performed on the thymus of Lck-FADD mice and littermate control. Scale bar: 20 μm. (g) NKAP western blots were performed on WT and FADD^−/− MEFs or Jurkat cells. Data are representative of three independent experiments. (h) MEF cells were transfected with a NKAP-specific siRNA and the expression of NKAP and Notch1 were measured using Q-PCR and western blotting. The data shown are from three independent experiments. *P<0.05, ***P<0.001