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Finally, GC B-cells exhibited significantly higher IFI16 mRNA levels than PCs (p can also be regulated posttranscriptionally [57], its expression, at the protein level, was analyzed in lymph nodes using an indirect immunofluorescence assay. Double staining with antibodies directed against IFI16 and the B-cell marker CD20 clearly demonstrated IFI16 protein in the lymph node compartments. As expected, there was strong CD20 staining in the germinal center and in the mantle zone, and IFI16 was detectable inside the nucleus of CD20 positive B-cells (Figures 2(a)�C2(c)). In accordance with the gene expression results, IFI16 staining was clearly brighter in the mantle zones, which are normally populated by na?ve and memory cells, than in the GCs, which are basically composed of centroblasts and centrocytes (Figure 2(c)). Figure 2 IFI16 protein expression in normal lymphoid tissues. (a) IFI16 staining of GC and mantle zones. (b) Double staining with CD20 and IFI16 antibodies. IFI16 protein expression in reactive lymphoid follicles; the expression pattern was largely restricted ... Because the mantle zones can be quite heterogeneous in their composition, we also evaluated IFI16 expression in na?ve and memory B-cell subsets purified from healthy donor peripheral blood samples using immunomagnetic procedures. Using flow cytometry, slightly higher IFI16 expression levels in memory cells compared with na?ve B-cells were detected (Figure 3), although the difference was not significant. Western blot analysis of protein extracts from peripheral blood na?ve and memory B-cell subsets demonstrated the presence of the three IFI16 isoforms generated by alternative splicing (Figure 3). Consistently, qPCR assays demonstrated that the IFI16 mRNA content was similar in peripheral blood na?ve and memory B-cell subsets (Figure 3), thus confirming the lymph node observations. Taken together, these results indicated that IFI16 expression was modulated Sitaxentan throughout B-cell differentiation with a progressive downregulation during the GC reaction and differentiation to PCs. Figure 3 IFI16 protein and mRNA expression analysis in na?ve and memory B-cells purified from peripheral blood. In (a), IFI16 mRNA levels were determined using qRT-PCR. The IFI16 mRNA expression relative quantification was calculated with the ����C ... 3.2.