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. Author manuscript; available in PMC: 2014 Oct 24.
Published in final edited form as: Cell. 2013 Oct 10;155(3):10.1016/j.cell.2013.09.049. doi: 10.1016/j.cell.2013.09.049

Figure 2. Phosphorylation of S366 of STING Inhibits IRF3 but not NF-κB Activity.

Figure 2

(A and B) HEK293T cells were transfected with plasmids encoding the luciferase gene under control of the indicated promoter with STING variants. After 36 hr, luciferase activity was measured.

(C) Primary STING−/− MEF cells were reconstituted with mSTING variants using retroviruses. The cells were transfected with dsDNA (4 µg/ml) for 3 hr and then stained with the indicated antibodies.

(D) Reconstituted STING−/− MEF cells were transfected with dsDNA (4 µg/ml) for the indicated times and immunoblots performed.

(E) Reconstituted STING−/− MEF cells were transfected with dsDNA (4 µg/ml) for 3 hr. RNA was purified and examined for gene expression with Illumina Sentrix BeadChip Array (Mouse WG6 version2). Pseudo colors indicate transcript levels below, equal to, or above the mean (green, black, and red, respectively). The scale represents the intensity of gene expression (log10 scale). The results shown here are representative of three independent experiments.

(F) Realtime PCR was carried out with the indicated probes to confirm gene array analysis shown in Figure 2E. Total RNA was extracted from reconstituted STING−/− MEF cells after dsDNA treatment for 3 hr and then cDNA was synthesized.

(G) The expression of NF-κB target genes in STING−/− MEF with S365D was comparable with that in STING−/− MEF with mSTING. Promoter sequence of listed genes (−1000 to +200) was obtained through DBTSS (http://dbtss.hgc.jp) and analyzed by TFSEARCH (http://www.cbrc.jp/research/db/TFSEARCH.html) at threshold score 85. The results shown here are the average of three independent experiments.

Asterisks indicate significant difference (P < 0.05) determined by Student’s t-test. ns means not significant. Error bars indicate sd. See also Figure S2.