PMC

a. Schematic of hSTING indicating transmembrane and leucine rich regions. b. Immunoblot analysis of STING in HEK 293 cells treated with RNAi to STING (hSTING) or control RNAi (NS). c. Northern blot analysis of human STING. d. Confocal analysis of HEK 293 cells transfected with hSTING tagged at the carboxyl end with HA. Transfected cells were also analyzed using ER-dsRed, Mitotracker or Golgi-dsRed. e. Fractionation experiments confirm that STING resides in the ER. Control antibodies indicate accuracy of fractionation (Calreticulin- ER, COX IV-mitochondria, β-actin- cytosol).

a. 293T cells were transfected with an IFNβ-Luc (p110-Luc) plasmid and increasing amounts of human (hSTING), murine (mSTING) or control ΔRIG-I. b. 293T cells transfected as in (a) with either PRD-III-I-Luc (b), NF-κB-Luc (c) or ISRE-Luc (d) reporter plasmids were analyzed similarly. e. MEF’s were transfected as in (a) and IFNβ mRNA was analyzed by qRT-PCR. f. Medium from transfected MEFs was analyzed for IFNβ protein by ELISA. g. Microarray analysis of 293T cells transfected with hSTING. h. MEF’s transfected with STING or ΔRIG-I or IPS-1 are resistant to VSV-GFP infection (MOI 1). i. Plaque assay from (h). j. TBK-1 deficient MEFs do not facilitate STING signaling. k. Schematic of hSTING variants. l. 293T cells were transfected as in (a) with hSTING or variants and luciferase measured. m. 293T cells were transfected with STING and increasing amounts of hSTING-Full, hSTING-N or hSTING-C with luciferase plasmids as in (a). Asterisks indicate significant difference (P < 0.05) as determined by Student’s t-test. Error bars; +/− s.d.

a. qRT-PCR analysis of mSTING mRNA in STING −/− or control MEFs. b. Immunoblot of mSTING in −/− cells or controls. c. Fluorescence microscopy (GFP) of mSTING −/− or controls infected with VSV-GFP (MOI 0.1). d. Viral titers from (c). e. Viral titers following VSVΔM infection. f. Endogenous IFNβ levels from STING −/− or controls after infection with VSV-GFP (MOI 1) or Sendai Virus (SeV MOI 1) g. STING −/− MEF’s or controls were treated with transfected B-DNA and IFNβ measured by ELISA. h. Time course analysis of (g). i. STING −/− or controls were exposed to transfected B-form DNA, interferon stimulatory DNA (ISD), or ISD reversed nucleotide sequene (ISDR), Listeria monocytogenes (MOI 50), HSV (MOI 5) for 12 hours, and IFNβ measured by ELISA. Asterisks indicate significant difference (P < 0.05) as determined by Student’s t-test. Error bars; +/− s.d. ND; not detectable.

a. 293T cells were co-transfected with HA-STING, FLAG-RIG-I or MDA5 and infected with SeV (MOI 1). Lysates were immunoprecipitated (IP) and immunoblotted (IB) using antibodies to HA or FLAG. b. Endogenous hSTING associates with RIG-I in HUVECs. c. ΔRIG-I (aa1-284) and not RIG-I-C (aa218-925) associates with STING in co-transfected 293T cells. d. Confocal image of 293T cells co-transfected with tagged STING and RIG-I. e. 293T cells were co-transfected with control vector (−) or increasing amounts of full-length, amino (aa1-230) or carboxyl (aa173-379) STING and ΔRIG-I, and IFNβ-Luc was measured. f. Control or STING −/− MEFs were transfected with ΔRIG-I (aa1-284) or IPS-1 and IFNβ was measured by ELISA. g. BD-hSTING-C interacts with Ssr-2/TRAPβ (AD-hTRAPβ) in yeast-two hybrid screening (BD-hSTINGΔSP, amino acids 36-369; BD-hSTING-C amino acids 173-379). h. HEK 293 cells were transfected with FLAG-tagged TRAPβ and endogenous STING or Sec61β measured by immunoblot. i. STING and TRAPβ were co-transfected HEK 293 cells and analysis carried out as in (h). j. Co-localization of STING and TRAPβ in 293T cells. k. RNAi to TRAPβ, SEC61β or Sec5 in HEK 293 cells ablates STING signaling. l. HA-STING associates with GFP-TBK-1 in co-transfected HEK 293 cells. Asterisks indicate significant difference (P < 0.05) as determined by Student’s t-test. Error bars; +/− s.d.