Immunoprecipitation of STING/ADSL and mass spectrometry analysis

BT-549 cells with or without expression of Flag-STING were treated with or without hypoxia for 6 h. An immunoprecipitation assay was performed using anti-Flag antibody, and immunoprecipitates of Flag-STING were eluted with Flag peptide, separated using SDS-PAGE and stained with Coomassie Brilliant Blue. Selected peptide hits of proteins associated with Flag-STING identified through mass spectrometry are shown.Bacterially purified His-ADSL proteins on Ni-NTA agarose beads were incubated with or without active GST-IKKβ in the presence of ATP for an in vitro kinase assay;Then the protein samples were digested using GluC and analyzed using LC-MS/MS with the Orbitrap Elite mass spectrometer.