Deletion of the RAM domain inhibits Notch4 nuclear localization. (A) Structure diagrams of the HA epitope-tagged Notch4 intracellular region (N4IC) and related deletion constructs. The amino acid (aa) numbers from the 2003 residue human Notch4 protein that are included in each mutant are indicated in parentheses. NLS indicates nuclear localization signal. (B) Expression of the N4IC constructs in HMECs as detected by immunoblotting with anti-HA antibody. (C) Expression and subcellular localization of the N4IC constructs as detected by immunofluorescent staining of transduced HMEC lines with anti-HA antibody. Nuclei are counterstained with DAPI. Original magnification, × 400; bar = 10 μm.