IP STING - Identification of new partners
To access STING function in absence of inflammation, given the tight interconnexion between metabolic and immune pathways, we questioned the impact of Sting ablation on metabolic parameters in vivo. Mice deficient for STING presented profound modifications of glucose management and body temperature. To identify the molecular mechanism through which Sting regulates metabolic homeostasis, we performed tandem-affinity purification of Flag- and HA-tagged Sting (F/HA-Sting) stably expressed in mouse embryonic fibroblasts (MEF) knockout for Sting (MEF Sting-/- ). Immunopurified material was analyzed by Mass Spectrometry to identify Sting protein partners.
Steps to reproduce
MEF Sting-/- overexpressing F/HA- Sting were lysed in 5 packed cell volume of TENTG-150. The first immunoprecipitation used an anti-FLAG antibody, followed by the elution using an excess of FLAG peptide (Sigma). Eluates were subsequently used as input material for immunoprecipitation using an anti-HA antibody. Sting protein partners were eluted using an excess of HA peptide (Thermo Scientific). Part of the FLAG and HA immunoprecipitated material was silver-stained and the remainder Coomassie-stained. Portions of the Coomassie-stained gel were excised and analyzed by Mass Spectrometry.