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. Author manuscript; available in PMC: 2021 Sep 25.
Published in final edited form as: Nat Immunol. 2021 Mar 25;22(4):485–496. doi: 10.1038/s41590-021-00896-3

Fig. 7 |. DAPK3 phosphorylation of LMO7 is necessary for STING K63-linked poly-ubiquitination.

Fig. 7 |

a, In vitro kinase assay of GST-tagged human LMO7 (aa 810-910). Peptides were incubated with GST-tagged DAPK3 or TBK1 in the presence of [γ-32P] ATP. b, (Upper) Immunoprecipitation and immunoblot of HEK293T transduced with indicated shRNA prior to transfection with plasmids encoding HA-tagged human STING and V5-tagged human LMO7(WT) and (lower) immunoblot of whole cell lysates (WCL). c, (Upper) Immunoprecipitation and immunoblot of HEK293T transfected with plasmids encoding HA-tagged human STING and V5-tagged human LMO7(WT) or phosphor-deficient LMO7 (S863A) and (lower) immunoblot of WCL. d, (Upper) Immunoprecipitation and immunoblot of HEK293T transfected with plasmids encoding 3×Flag-tagged human STING, HA-tagged Ub(K63O), and V5-tagged human LMO7 mutants, and (lower) immunoblot of WCL. e, Schematic representation of human LMO7 mutants. f, g, (Upper) Immunoprecipitation and immunoblot of THP1-Blue ISG transduced with two distinct (f) shLMO7 or (g) shTRIP12 and stimulated with 2′,3′-cGAMP (10 μg/ml) for 3 h and 6 h, and (lower) immunoblot of WCL. Endogenous K63-linked poly-ubiquitin chains were immunoprecipitated using K63-TUBE-Flag. h, i, qRT-PCR analysis of IFNB1 and CXCL10 in THP1-Blue ISG transduced with two distinct (h) shLMO7 sequences or (i) shTRIP12 sequences and stimulated with VACV70 (4 μg/ml), 2′,3′-cGAMP (10 μg/ml), or c-di-GMP (10 μg/ml) for 4 h. Data in (a-d, f, g) are representative of or (h, i) the mean of three independent experiments. Values represent mean ± s.d. *P<0.05, **P<0.01, and ***P<0.001. Statistical comparisons were conducted using two-tailed t-test (h, i).