Fig. 2. The full-length (150 kDa) form of hADAR1 localizes to the cytoplasm and the short (110 kDa) form localizes to the nucleolus. (A) Schematic representation of differently tagged hADAR1 constructs. In-frame methionines present in hADAR1 cDNA are shown (M296, M337). The NLS recently described by Eckmann et al. (Eckmann et al., 2001) is marked by an asterisk (*). (B) HeLa cells were transiently transfected with the indicated hADAR1 constructs and assayed for ADAR1 localization by either direct detection of GFP or indirect immunofluorescence using anti-FLAG and anti-His antibodies. GFP- and FLAG-tagged ADAR1 localize to the cytoplasm. The anti-His antibody stains both the cytoplasm and the nucleolus. In addition, the anti-His antibody labels discrete aggregates in the nucleoplasm (arrow). (C) Western-blot analysis of total proteins from COS7 cells transfected with the indicated plasmids. Total cell extracts after 36 hours of expression were prepared and fractionated by SDS-PAGE. Western blotting was then performed with an anti-GFP antibody (lanes 1, 2), an anti-FLAG antibody (lanes 3, 4) and an anti-His antibody (lanes 5-7). The western-blot signal observed with mobility just below that of the ADAR1 band with the anti-FLAG antibody is a nonspecific signal also present in non-transfected cells (Fig. 2C, lanes 3,4). Both antibodies recognize the full-length (150 kDa) hADAR1 but the short (110 kDa) form of the protein is only specifically detected with anti-His antibody. Molecular weight markers are shown on the left.