Expression of Irga6 in Liver Figure 2. Irga6 is expressed in many mouse tissues. A. Western blot analysis of Irga6 protein expression. Detergent lysates from liver, heart, skin, spleen, intestine, thymus, lung, testis, kidney and brain of wild-type C57BL/6 mice were probed for Irga6 protein using mAb 10D7. Loading for all tissues was 50 mg/lane except for skin, which was 150 mg/lane. L929 mouse fibroblasts (5,000 cells/lane) untreated (L929 IFN-) or stimulated (L929 IFN+) with 200 U/ml IFNc for 24 h were used as negative and positive controls. Calnexin protein was probed as loading control. An endoproteolytic cleavage product of calnexin is apparent in some lanes, especially in the Heart and Kidney samples. B. Immunohistochemical analysis of Irga6 protein expression. C57BL/6 WT and Irga62/2 adult mouse kidney, heart, spleen and thymus were embedded in paraffin and cut into 6 m sections. Irga6 protein was probed with mAb 10D7 and detected histochemically to give a green stain. Nuclei were counterstained in red. Frames and arrows show enlarged sections. doi:10.1371/journal.pone.0006787.g002 1A and 1B are separated by 6558 bp on chromosome 18 and the long coding exon 2 begins 7018 bp downstream of the 39 margin of exon 1B. Definition of the transcriptional start sites for exons 1A and 1B (Figure S1, see also above) allowed for the identification of putative transcription factor binding sites. In our previous analysis 5 IFN response elements were found upstream of exon 1A, including 2 ISREs and 3 GAS sequences, and single GAS and ISRE sequences upstream of exon 1B (Bekpen et al, 2005). A search for liver specific response elements in the 1000 bases upstream of exons 1A and 1B now revealed that exon 1B is preceded by an apparently canonical liver-specific promoter, while no liver response elements were found closer than 900 bases upstream of exon 1A (Fig. 4A). Alignments of the 7 hepatocyte nuclear factor (HNF) promoter elements found in the 1B promoter with the consensus sequence for each element are shown in Fig. 4B. Not only the identity of the HNF elements, but also their array on the putative promoter conform to the pattern of typical liver- specific promoters [17,18]. PLoS ONE | www.plosone.org 4 August 2009 | Volume 4 | Issue 8 | e6787