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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 3. ULK1 Negatively Regulates IFNβ Production by Phosphorylating STING S366.

Figure 3

(A) ELLISGMEK which includes Ser366 was used as substrate to identify ULK1 and ULK2.

(B) Schematic of human ULK1. KD is kinase domain.

(C) In vitro kinase assay was performed with recombinant ULK1 protein using recombinant hSTING protein as substrate. The indicated serine sites that were identified by mass spectrometry were substituted with alanine.

(D) Recombinant hSTING protein was incubated with recombinant ULK1 in presence of ATP for 15 min. Phosphorylated STING was analyzed by mass spectrometry. Highlighted amino acid (S366) was identified as the only phosphorylation site.

(E and F) Primary MEF cells (E) or hTERT-BJ1 cells (F) were treated with siRNA as indicated (NS: nonspecific siRNA) and then transfected with dsDNA (4 µg/ml) for 16 hr. IFNβ was measured by ELISA. Knockdown efficiency of ULK1 in primary MEF cells was confirmed by immunoblot.

(G) siRNA-treated primary MEF cells were infected with HSV-1 (MOI = 0.1) for 24 h and then plaque assay was performed.

(H) siRNA-treated hTERT-BJ1 cells were transfected with dsDNA (4 µg/ml) for the indicated times and then immunoblot was performed.

Asterisks indicate significant difference (P < 0.05) compared to NS determined by Student’s t-test. ns means not significant. Error bars indicate sd. See also Figure S3.