Figure 2.

PS1 potentiates nuclear translocation of the Notch-1 intracellular domain. PS1-wt and PS1-KO cells were transfected with Myc-tagged NδEF. The cells were fixed 48 hr after transfection, and immunofluorescence microscopy was performed with monoclonal anti-Myc. (A) PS1-wt cells show predominantly nuclear immunoreactivity. (B) PS1-wt cell that shows prominent nuclear immunoreactivity and lower levels of cytoplasmic staining. (C) PS1-KO cells show cytoplasmic immunoreactivity in a vesicular distribution; most cells do not show nuclear immunoreactivity. (D) The cleaved Notch-1 fragment is detected in the nucleus of PS1-wt but not PS1-KO cells. Nuclear and membrane fractions were prepared from NδE-transfected PS1-wt and PS1-KO cells, and the distribution of cleaved and uncleaved Notch-1 proteins was determined by immunoblot analysis. Note that in PS1-wt cells, the cleaved Notch-1 fragment is detected in the nuclear extract but not in the membrane fraction, whereas uncleaved NδE is present in the membrane fraction. In PS1-KO cells, the cleaved fragment is not detected at significant levels in the nuclear extract, but appears after transfection of PS1. The cleaved fragment is not restored in the nuclear extract after transfection of the PS1 mutant C410Y. The difference in cytoplasmic membrane immunoreactivity for NδEF in A and B vs. NδE in D is due to the increased cleavage of the NδEF construct (Fig. 1D).