Fig. 7: Ubiquitination on K288 of STING is required for STING degradation and termination of type I interferon response. | Nature Cell Biology

Fig. 7: Ubiquitination on K288 of STING is required for STING degradation and termination of type I interferon response.

From: STING signalling is terminated through ESCRT-dependent microautophagy of vesicles originating from recycling endosomes

Fig. 7

a, Sting−/− MEFs reconstituted with EGFP-STING were stimulated with DMXAA for the indicated times. EGFP-STING was immunoprecipitated with anti-GFP antibody. The cell lysates and the immunoprecipitated proteins were analysed by western blot. IP, immunoprecipitation. b, Sting−/− MEFs stably expressing mRuby3-STING and mNeonGreen (mNG)-ubiquitin were imaged every 5 min after DMXAA stimulation. c, Quantitation of the number of mNG-ubiquitin puncta (see also Supplementary Video 3). d, Sting−/− MEFs reconstituted with EGFP-STING (WT, K19R, K150/151R, K235R, K288R or K337R) were stimulated with DMXAA. EGFP-STING was immunoprecipitated with anti-GFP antibody. The cell lysates and the immunoprecipitated proteins were analysed by western blot. e, The fluorescence intensity of EGFP-STING (WT or K288R) under the indicated conditions was quantified. NS, not significant. f, Sting−/− MEFs reconstituted with EGFP-STING (WT or K288R) were stimulated with DMXAA. Cells were immunostained with anti-GM130 or anti-Rab11 antibodies. The Pearson’s correlation coefficient between EGFP-STING (WT or K288R) and GM130, or between EGFP-STING (WT or K288R) and Rab11, is shown. g, Cells were stimulated with DMXAA. Cell lysates were analysed by western blot. The band intensities were quantified. [STING/tubulin], [pTBK1/TBK1] and [pIRF3/IRF3] were calculated. h, Cells were stimulated with DMXAA or HT-DNA for 12 h. The expression of Cxcl10 was quantitated with qRT–PCR. Data are presented as mean ± s.d. i, Sting−/− MEFs reconstituted with EGFP-STING (WT or K288R) were stimulated with DMXAA. Cells were immunostained with anti-K63 ubiquitin antibody. j, The Pearson’s correlation coefficient between EGFP-STING (WT or K288R) and K63 ubiquitin is shown. k, Cells were stimulated with DMXAA. Cell lysates were prepared, and EGFP-STING was immunoprecipitated with anti-GFP antibody. The cell lysates and the immunoprecipitated proteins were analysed by western blot. Scale bars, 10 µm (b,f,i) and 500 nm (magnified images in b and i). Data are presented in box-and-whisker plots with the minimum, maximum, sample median and first versus third quartiles (e,f,j). The sample size (n) represents the number of cells (e,f,j) or the biological replicates (h). Source numerical data and unprocessed blots are available in source data.

Source data

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