Fig. 7. hADAR2 does not shuttle and an N-terminal domain is responsible for its nuclear localization. (A) Schematic representation of the GFP-tagged full-length hADAR2 and the GFP-ADAR2 deletions constructed to study putative localization signals in hADAR2. Numbers denote amino acid positions relative to the N-terminus of hADAR2. Asterisks indicate putative NLSs identified by sequence analysis, and the respective amino acid sequences are shown. (B) HeLa cells were transiently transfected with the indicated GFP-ADAR2 constructs. Approximately 16 hours after transfection, cells were fixed and directly observed with the fluorescence microscope. (C) HeLa cells were transfected with GFP-ADAR2 and fused with murine NIH 3T3 cells. The heterokaryons were incubated in the presence of emetine. Immunostaining with anti-hnRNP C was used as control. Both GFP-ADAR2 and hnRNP C molecules remain restricted to the transfected HeLa nucleus. The dashed lines indicate the contour of the murine nucleus in the heterokaryon. Incubation of HeLa cells expressing GFP-ADAR2 at 4°C in the presence of emetine shows that the protein does not leak to the cytoplasm. Bar, 10 µm.