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. 2014 May;88(10):5328–5341. doi: 10.1128/JVI.00037-14

FIG 3.

FIG 3

TBK1 regulates dsDNA-mediated NF-κB activation in MEFs. (A) Immortalized MEFs derived from wild-type (TBK1+/+) or TBK1-deficient (TBK1−/−) mice were stimulated with 10 μg/ml of dsDNA90 or 5 μg/ml of poly(I·C) for the indicated times. The expression levels of TBK1, STING, IRF3 phosphorylated at Ser396 (p-IRF3), NF-κBp65 phosphorylated at Ser536 (p-NF-κBp65), NF-κBp65, and β-actin were determined by immunoblotting. (B) TBK1+/+ and TBK1−/− MEFs were stimulated with 10 μg/ml of dsDNA90 or 5 μg/ml of poly(I·C) for 3 h, and then the cells were stained with antibodies against NF-κBp65 or IRF3. (C) Detection of nuclear translocation of NF-κBp65 by fractionation assay. TBK1+/+ and TBK1−/− MEFs were stimulated with 10 μg/ml of dsDNA90 or 5 μg/ml of poly(I·C) for 3 h, and then cell lysates were separated into cytosolic and nuclear fractions. Each fraction was concentrated and subjected to immunoblotting with the indicated antibodies. (D) Quantitative analysis of NF-κBp65 phosphorylated at Ser536 by ELISA. TBK1+/+ and TBK1−/− MEFs were stimulated with 10 μg/ml of dsDNA90 or 5 μg/ml of poly(I·C) for 3 h, and then endogenous levels of NF-κBp65 phosphorylated at Ser536 were determined by ELISA. *, P < 0.05 (Student's t test). Error bars indicate SD. OD, optical density.