Fig. 3

Bacterial nucleic acids stimulate IRF3 phosphorylation in hμglia human microglial cells. Cells were transfected with 0.1 μg/ml BDNA, 1 μg/ml 5′pppRNA, 0.5 μg/ml bacterial genomic DNA (gDNA) (a), or 1 μg/ml bacterial RNA (b). Cell lysates were collected at 1, 2, and 3 h and analyzed for protein expression of phosphorylated IRF3 (pIRF3) and the housekeeping gene, β-actin, via immunoblot analysis. Relative phosphorylated IRF3 normalized to β-actin is displayed graphically (a, b). Additionally, at 3 h post transfection, the cytosolic and nuclear cells fractions were separated and analyzed for protein expression of IRF3 and β-actin via immunoblot analysis (c). Relative IRF3 protein expression was normalized to β-actin and is displayed graphically below the representative immunoblot. Data are expressed as mean ± SEM for a minimum of three independent experiments. Asterisks indicate statistical significance compared to unchallenged cells as determined by two-way ANOVA (p < 0.05)