Fig. 2. Alternative splicing and 3'ss selection are affected by hSlu7 nuclear concentration. (A) The ADAR2 minigenes and the sequence of the Alu exon 3'ss. The selected 3'ss is underlined. Proximal (left) and distal (right) AGs are indicated in bold. The 3'ss of the intron-2/exon-3 junction of the gene encoding D-aspartate oxidase (DDO) is shown at the bottom. Lower- and upper-case letters represent intron and exon sequences, respectively. (B) Cells were transfected with either of the ADAR2 mini-genes (ADAR2 and ADAR2+10) (Lev-Maor et al., 2003). Forty-eight hours after transfection, cells were either mock treated or UV-C irradiated and then incubated for 3 hours at optimal conditions. Then, cytoplasmic RNA was collected followed by RT-PCR using specific primers and separation on 2% agarose gel (lanes 2-9). Some of the cells were co-transfected with a plasmid for RNAi directed against hSlu7 (RNAi) or a control plasmid (Plasmid; lanes 6-9). A schematic representation of PCR products is indicated to the right of the gel. The alternative Alu exon is colored black. GAPDH, a house-keeping gene, was amplified from each sample to confirm that approximately the same amount of cDNA was used for each reaction. (C) A graph quantifying the spliced products, including exon 8 from B, expressed out of the total amount of spliced products in the same lane and experiment. The error bars represent s.d. (D) Cells were transfected with either an RNAi plasmid directed against hSlu7 or a control plasmid. After 1 day or 2 days, total proteins were extracted, separated by 12% SDS-PAGE, transferred to a membrane and probed for hSlu7, hPrp18 and GAPDH. Lane 1: `No trans.' indicates cells that were not transfected. Lane 2: `Plasmid' indicates cells transfected with a control plasmid. Lanes 3 and 4, `RNAi' indicates cells transfected with an RNAi plasmid directed against hSlu7 for 1 day (1d) or 2 days (2d), respectively. The 55 kDa protein is a non-specific interaction of anti-hPrp18 antibody. (E) Cells were either mock treated (lane 2) or UV-C irradiated (lane 3), then incubated for 3 hours at optimal conditions followed by RNA isolation and RT-PCR. Some of the cells were transfected with an RNAi plasmid directed against hSlu7 (RNAi; lane 5) or a control plasmid (Plasmid; lane 4). A schematic representation of PCR products is indicated to the right of the gel. (F) A graph quantifying the spliced products including/excluding the alternative DDO exon from E expressed out of the total amount of spliced products in the same lane and experiment.