Fig. 1. Subcellular distribution of endogenous ADAR1. (A) Schematic representation of hADAR1 proteins (150 kDa and 110 kDa forms). The most important domains of hADAR1 are indicated as follow: ZBDs, Z-DNA-binding domains (light gray); dsRBDs, double-stranded RNA binding domains (black boxes); deaminase domain (dark gray box). The partial ADAR1 fragments (black lines) shown were expressed in E. coli and used for production of rabbit polyclonal antibodies. Numbers denote amino acid positions relative to full-length hADAR1. (B) Western-blot analysis of HeLa and COS7 total cell extracts prepared and fractionated by SDS-PAGE. Western blotting was then performed with a pre-immune serum (lane 1) and with anti-ADAR1 antibodies: 007 (lane 2) and 668 (lane 3-5). Extracts of COS7 cells transiently transfected with Flis-ADAR1 (Fig. 2) are shown in lane 5. All antibodies recognized the full-length hADAR1 (150 kDa) and the 110 kDa form of ADAR1. Molecular weight markers are shown on the left. (C) HeLa cells were immunostained with antibodies 007 and 668, and with the respective pre-immune sera. The panels are representative of the labeling patterns observed. (D) Neurosecretory neurons from the supraoptic nucleus were obtained from squash preparations of rat hypothalamus and analysed by indirect immunofluorescence. The cells were double-labeled with affinity-purified anti-ADAR1 antibody (007) and an anti-histone antibody. The ADAR1 antibody produces a non-homogeneous nucleoplasmic staining, with additional labeling of the nucleolus (arrow). Within the nucleoplasm, the antibody decorates nuclear speckles. Bar, 10 µm.