Fig. 9. Nuclear staining of NRADD requires -secretase. (A) N2a cells were treated with APMA or PMA for 40 minutes, washed and incubated for 3 hours in lactacystin and DAPT where indicated. Cells were fixed, immunostained with anti-NRADD antibody and visualized by confocal microscopy. Notice the diffuse nuclear staining in the presence of lactacystin alone. NRADD containing nuclear substructures are induced by treatment with APMA or PMA (arrows). Co-incubation with the -secretase inhibitor DAPT prevents the formation of these structures. (B) A series of confocal pictures show that the nuclear substructures localize within the DAPI-stained nuclei but do not localize with high-intensity DAPI subnuclear structures.