Expression of Irga6 in Liver Figure 1. Irga6 is constitutively expressed in healthy mouse liver. A. Northern blot analysis of Irga6 RNA expression. Total RNA was extracted from livers of wild-type C57BL/6J or IFN-c receptor knock out (IFN-cR2/2) mice, which were either untreated (2) or had been infected with Listeria monocytogenes at LD50 24 h earlier (+). The Irga6 ORF labeled with [a-32P]-dCTP was used as probe. L929 mouse fibroblasts, either unstimulated (2) or stimulated (+) with 200 U/ml IFNc for 24 h, were used as controls. GAPDH was also detected as loading control. The y-axis of the histogram presents relative units of Irga6 signal normalized to GAPDH. B. Western blot analysis of Irga6 protein expression. Detergent lysates of livers prepared as described in Materials and Methods (100 mg/lane) from CB20 baby (3 or 4 days, 3 individuals) and adult mice (4 or 6 weeks, 4 individuals) were probed for Irga6 protein with mAb 10D7. L929 mouse fibroblasts (5,000 cells/lane) stimulated with 200 U/ml IFNc for 24 h were used as positive control. Calnexin protein was probed as loading control. An endoproteolytic cleavage product of calnexin is apparent in some lanes, especially Lane 2. C. In situ hybridization analysis of Irga6 RNA expression. Livers from adult C57BL/6 mice were fixed, embedded in paraffin and cut into 6 m sections. DIG-labeled single-stranded RNA sense and anti-sense probes were synthesized by in vitro transcription using the 59 end of the Irga6 ORF (12490 bp) as template. The hybridized RNA probes were detected with anti-DIG antibody to give a blue stain. For details see Materials and Methods. D. Immunohistochemical analysis of Irga6 protein expression. Paraffin sections of livers from adult C57BL/6 wild-type (WT) and Irga62/2 mice were probed for Irga6 protein with rabbit anti-Irga6 antiserum 165/3. Specific antibody binding was detected histochemically as described in Materials and Methods to yield a green stain. Nuclei were counterstained in red. doi:10.1371/journal.pone.0006787.g001 and exon1B in tandem with a short spacer between them (NM_001146275). This, till now unique, structure is unlikely to be of any biological significance. Fig. 4A shows diagrammatically the intron-exon structure of the Irga6 gene and the organization of the 1A and 1B promoters (see also Fig. 5 and Table S2). In the genomic structure of Irga6 exons PLoS ONE | www.plosone.org 3 August 2009 | Volume 4 | Issue 8 | e6787