Figure 7.
N1 and R848 synergistically activate NF-κB, IRF3, and IRF7 signaling as well as type 1 IFN responses in mouse BMDCs. (A–C) Mouse BMDCs (2 × 106 cells/ml) were treated for 0–90 min with N1 (500 ng/ml) and R848 (500 ng/ml) before they were solubilized in the buffer for analysis of the translocation of proteins from cytoplasmic to nuclear cell fractions. An identical amount of proteins was loaded and separated by electrophoresis to detect the levels of cytoplasmic fraction of IRF3, IRF7, phospho-p65, and GAPDH, as well as nuclear fraction of IRF3, IRF7, phospho-p65, and lamin B1. Quantitation of band intensities from three different batches of male mice (n = 6) were normalized against GAPDH or lamin B1 using ImageJ software. (D) Mouse BMDCs were treated with N1 (500 ng/ml) and/or R848 (500 ng/ml) for 6 h before being used for the extraction of total RNA. The levels of IFN-β1 mRNA was quantitated by qPCR and shown as fold increase over the sham treated DCs. Data are shown as the average (mean ± SD) of triplicates of one experiment representative of three. *p<0.05 according to one-way ANOVA followed by Tukey's post hoc test.