Expression of Irga6 in Liver Figure 6. Constitutive and induced expression of Irga6 in mouse primary hepatocytes. A. Western blot analysis of Irga6 protein expression. C57BL/6 mouse primary hepatocytes were isolated as described in Materials and Methods. Protein lysates of liver or primary hepatocytes cultured and harvested at indicated hours (h) or days (d) post-isolation were probed for Irga6 protein with mAb 10D7 by Western blotting. Hepatocytes were cultivated with (24h+) or without (24h-) 200 U/ml IFN for 24 hours. L929 and TIB73 cells (5,000 cells/lane) stimulated with IFNc (200 U/ml) for 24 h were used as positive control. Calnexin protein was probed as loading control. B. Immunofluorescence analysis of Irga6 protein expression in hepatocytes. C57BL/6 mouse primary hepatocytes were isolated, cultured for 6 h, fixed and immunostained for Irga6 protein. Double staining using either Irga6 mAb 10E7 (green) and calnexin rabbit antiserum (red) (upper row) or Irga6 rabbit antiserum 165/3 (red) and anti-GM130 mAb (green) (lower row) were performed. Nuclei were counterstained with DAPI (blue). doi:10.1371/journal.pone.0006787.g006 hepatocytes. The levels of Irgm1, Irgb6 (Fig S3) and Irgd (not shown) were significant but below the expression levels in IFNg- induced fibroblasts. Irgm3 was barely detectable above back- ground (Fig S3 and data not shown). All of these proteins were strongly inducible in primary hepatocytes by IFNg (Fig S3 and data not shown) Immunofluorescence analysis of the expression of Irga6 in freshly isolated primary hepatocytes confirmed the high level of expression (Fig. 6B). The Irga6 signal accurately co-localised with the signal for the ER protein, calnexin. No significant expression of hepatocyte Irga6 was apparently associated with the distributed Golgi elements of these cells. Accumulation of Irga6 on the parasitophorous vacuoles of T. gondii infecting isolated primary hepatocytes As previously reported, Irga6 and other IRG proteins accumulate rapidly on the parasitophorous vacuoles of infecting T. gondii in IFNc-induced cells [11,22]. We were interested to know whether the constitutively expressed Irga6 in primary hepatocytes could also accumulate on the T. gondii PV. Freshly isolated primary hepatocytes were therefore infected with T. gondii strain ME49 and Irga6 distribution was examined by immunofluorescence. Intense ring-shaped fluorescence of Irga6 surrounding some infecting T.gondii was observed (Fig. 7A). It was, however, also noted that the proportion of Irga6-coated T. PLoS ONE | www.plosone.org 8 August 2009 | Volume 4 | Issue 8 | e6787