FIG 8 .

IFI205 binds to the cytoplasmic DNAs of endogenous retrotransposons and induces a cGAMP-independent IFN response (A) DNA pulldown assay for IFI205, AIM2, and cGAS. NIH 3T3 cells were transiently transfected with expression plasmids for IFI205myc, AIM2HA, or cGASV5 and harvested 48 h after transfection. The cytoplasmic fractions were subjected to a DNA pulldown assay with the antibodies indicated. Normal IgG was used as a negative control. Bound DNA copy numbers were measured by qPCR and normalized to input DNA values. The assays were repeated three times for each protein, and qPCR was performed in triplicate for each experiment. Protein expression was confirmed by Western blotting with the same lysates and antibodies used for the DNA pulldown. (B) NR9456 cells were transfected with the siRNAs indicated and cGAMP at the concentrations shown. IFN-β expression levels were measured by RT-qPCR and normalized to Gapdh. The values shown are the mean ± the standard error of the mean of three experiments. *, P < 0.05; **, P < 0.005; ***, P < 0.0005; NS, not significant (two-tailed t test). siCont, control siRNA.