IFI16 Expression Is Related to Selected Transcription Factors during B-Cell Differentiation

Figure 3

IFI16 protein and mRNA expression analysis in naïve 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 method [34, 35]. The results are shown for the naïve B-cell subset relative to the memory B subset. The data represent the mean (±SD) of three independent experiments performed in duplicate. In (b), flow cytometry analysis of intracellular IFI16 protein was performed in naïve and memory B-cell subsets purified from peripheral blood. Naïve (light grey histogram) and memory (grey histogram) B-cells were stained by indirect immunofluorescence with a rabbit anti-IFI16 antibody (1 : 40 in 0.2% saponin/PBS) and, subsequently, with a FITC-conjugated sheep anti-rabbit IgG (1 : 100 in 0.2% saponin/PBS). The white histograms are the negative controls (dotted line, memory cells, solid line, and naïve cells) represented by naïve and memory B-cells stained with indirect immunofluorescence with a rabbit anti-HIV-1 p24 antibody (1 : 40 in 0.2% saponin/PBS) and, subsequently, with a FITC-conjugated sheep anti-rabbit IgG (1 : 100 in 0.2% saponin/PBS). A representative experiment is shown. In (c), western blot analysis of protein extract from peripheral blood memory and naïve B-cell subsets (). Cell lysates were separated by gel electrophoresis and transferred to nitrocellulose membrane. The proteins were probed with rabbit anti-IFI16 polyclonal antibody and then incubated with an AP-conjugated anti-rabbit IgG and detected by colorimetric procedure. Tubulin protein was assayed as control. IFI16 A, B, and C isoform proteins were expressed similarly in memory and naïve B-cell subsets. A representative experiment is shown.

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