Fig 1: SELENOK interacts with STING.(A) ELISA analysis of IFN-ß secretion in PMs from WT and Selenok-deficient mice stimulated with HSV-1 or ionomycin plus HSV-1. (B) Immunoblots of MYC immunoprecipitation in HEK 293T cells transfected with STING-MYC and the control vector or SELENOK-GFP plasmids. (C) Immunoblots of GFP immunoprecipitation in HEK 293T cells transfected with SELENOK-GFP and the control vector or STING-HA plasmids. (D) Immunoprecipitation using a STING-specific antibody in PMs stimulated with cGAMP. (E) Immunofluorescence analysis of PMs stimulated by cGAMP or not using anti-SELENOK (green) and anti-STING (red) antibodies and DAPI nuclear staining. Scale bar,10µm. The intensity profiles of each line were quantified by Image J software. (F) Immunofluorescence analysis of PMs stimulated by cGAMP or not using anti-SELENOK (green) and anti-GM130 (red) antibodies and DAPI nuclear staining. Scale bar,10µm. The intensity profiles of each line were quantified by Image J software. (G) Schematic of the SELENOK and its truncated body. (H) HA immunoprecipitation in HEK 293T cells transfected with STING-HA and the control vector, SELENOK-GFP, SELENOK-GFP N42, or SELENOK-GFP C43 plasmids. The specific bands were marked by asterisks. (I) Schematic of the STING and its truncated body. (J) GFP immunoprecipitation in HEK 293T cells transfected with SELENOK-GFP and the control vector, STING-HA, STING-HA C139. Statistical significance was determined by unpaired two-sided multiple Student’s t-tests in A. Data represent mean ± standard deviation (SD) or one representative image from three independent experiments. ns: not significant; ***P: < 0.001.
Fig 2: SELENOK enhances the oligomerization of STING.(A) Immunoblot analysis of STING dimerization in PMs stimulated by cGAMP. (B) Immunoblot analysis of oligomerization of STING in PMs stimulated by BFA, HSV-1, or BFA plus HSV-1. (C) Immunoblot analysis of oligomerization of STING in PMs stimulated by BFA, cGAMP, or BFA plus cGAMP. (D) Immunoblot analysis of SEC13 expression in PMs transfected with siRNA (siNC, siSec13-1 and siSec13-2). (E) Immunoblot analysis of oligomerization of STING in PMs stimulated by cGAMP after transfection with siRNA (siNC, siSec13-2). (F) Immunofluorescence analysis of MEF stimulated with cGAMP using anti-STING (green) and anti-COP-II (red) antibodies and DAPI nuclear staining. Scale bar,10µm. (G) Colocalization analysis of STING and COP-II by Manders’ Colocalization Coefficients (MCC). (H) Immunofluorescence analysis of MEF stimulated with HSV-1 using anti-STING (green) and anti-GM130 (red) antibodies and DAPI nuclear staining. Scale bar,10µm. (I) Colocalization analysis of STING and GM130 by Manders’ Colocalization Coefficients (MCC). (J) Immunoblot analysis of STING oligomerization in HEK 293T cells transfected with STING-HA and control vector plasmids or SELENOK-FLAG, followed by stimulation with cGAMP. (K) Immunoblot analysis of STING oligomerization in HEK 293T cells transfected with STING-MYC, the control vector or SELENOK-FLAG, and then stimulated by BFA or not. (L) Immunoblot analysis of STING palmitoylation in PMs stimulated by cGAMP. (M) Immunoblot analysis of STING oligomerization in HEK 293T cells transfected with STING-MYC, STING-MYC C88/91S, the control vector, and SELENOK-FLAG. Statistical significance was determined by unpaired two-sided multiple Student’s t-tests in G and I. Data represent mean ± standard deviation (SD) or one representative image from three independent experiments. **P: < 0.01, ***P: < 0.001. siNC, control siRNA; siSec13-1; siSec13-2 (S1 Table).
Fig 3: Selenok deficiency specifically attenuates cGAS-STING signaling.(A) ELISA analysis of IFN-ß secretion in PMs following stimulation with HSV-1, ISD, and SeV. (B) Real-time PCR analysis of Ifnb mRNA levels in PMs following stimulation with HSV-1, ISD, and SeV. (C) ELISA analysis of IL-6 secretion in PMs following stimulation with HSV-1 and ISD. (D-F) Immunoblot analysis of p-IRF3, p-TBK1, p-STAT1,and Viperin expression in PMs following stimulation with (D) HSV-1 or (E) ISD or (F) SeV. (G) Real-time PCR analysis of Cxcl10, Rantes, Isg15, and Isg54 mRNA levels in PMs following stimulation with HSV-1. (H) ELISA analysis of IFN-ß secretion in BMDMs following stimulation with HSV-1. (I) Real-time PCR analysis of Ifnb mRNA levels in BMDMs following stimulation with HSV-1. (J) ELISA analysis of IFN-ß secretion in MEFs following stimulation with HSV-1. (K) Real-time PCR analysis of Ifnb mRNA levels in MEFs following stimulation with HSV-1. Statistical significance was determined by unpaired two-sided multiple Student’s t-tests in A-C and G-K. Data represent mean ± standard deviation (SD) or one representative image from three independent experiments. ns: not significant, *P: < 0.05, **P: < 0.01, ***P: < 0.001.
Fig 4: Selenok deficiency inhibits STING activation.(A) Luciferase activity assays of IFN-ß activation in HEK 293T cells transfected with control vector or SELENOK, followed by transfection with control vector (Mock), cGAS plus STING, and TBK1. (B) Immunoblot analysis of phosphorylated and total STING in PMs stimulated with HSV-1. (C) In vitro pull-down of cGAS and ISD. (D) ELISA analysis of cGAMP secretion in PMs stimulated with HSV-1. (E) ELISA analysis of IFN-ß secretion in PMs following stimulation with cGAMP and DMXAA. (F) ELISA analysis of IL-6 secretion in PMs following stimulation with cGAMP and DMXAA. (G)Real-time PCR analysis of Ifnb mRNA levels in PMs stimulated by cGAMP and DMXAA. (H) ELISA analysis of IFN-ß secretion in BMDMs following stimulation with cGAMP and DMXAA. (I and J) Immunoblot analysis of p-IRF3, p-TBK1, and Viperin expression with (I) cGAMP stimulation or (J) DMXAA stimulation. (K) Real-time PCR analysis of Cxcl10, Rantes, Isg15, and Isg54 mRNA levels in PMs following stimulation with cGAMP and DMXAA. (L–M) Mice were stimulated by DMXAA for 6 h by i.p. injection. (L) Serum levels of IFN-ß were analyzed using ELISA (mock, n = 3; DMXAA, n = 5). (M) Serum levels of IL-6 were analyzed using ELISA (mock, n = 3; DMXAA, n = 5). Statistical significance was determined by unpaired two-sided multiple Student’s t-tests in A, D-H, and K-M. Data represent mean ± standard deviation (SD) or one representative image from three independent experiments. ns: not significant. *: P < 0.05, **P: < 0.01, ***P: < 0.001.
Fig 5: Selenok knockdown specifically inhibits cGAS-STING signaling.(A) Immunoblot analysis of SELENOK expression in PMs transfected with siRNA. (B) ELISA analysis of IFN-ß secretion in PMs stimulated with HSV-1, ISD, and SeV after transfection with siRNA. (C) Real-time PCR analysis of Ifnb mRNA levels in PMs stimulated with HSV-1, ISD, and SeV after transfection with siRNA. (D) Immunoblot analysis of p-IRF3, p-TBK1, p-STAT1, and Viperin expression in PMs stimulated with HSV-1 after transfection with siRNA. (E) Immunoblot analysis of p-IRF3, p-STAT1, and Viperin expression in PMs stimulated with ISD after transfection with siRNA. (F) ELISA analysis of IFN-ß secretion in BMDMs stimulated with HSV-1. (G) Real-time PCR analysis of Ifnb mRNA levels in BMDMs stimulated with HSV-1. Statistical significance was determined by unpaired two-sided multiple Student’s t-tests in B-C and F-G. Data represent mean ± standard deviation (SD) or one representative image from three independent experiments. ns: not significant, *: P < 0.05, **: P < 0.05, ***P: < 0.001. siNC, control siRNA; siSelenok-1,2,3, and mix (S1 Table).
Supplier Page from OriGene Technologies for STING (TMEM173) (NM_198282) Human Recombinant Protein