Ectopic protein expression in HEK293T cells

HEK293T cells were seeded into 6-cm dishes at ∼70% confluency. The cells were transfected with 2 µg FLAG-tagged-STING (Addgene) and 2 µg hemagglutinin (HA)-tagged ubiquitin (Addgene) and 2 µg NT3 (WT) or NT3 (ΔDUB) using Lipofectamine 2000 (Invitrogen). After 24 h of transfection, cells were lysed in IP lysis buffer containing 1× cOmplete mini, EDTA- free protease inhibitor (Sigma), 1× PhosSTOP (Sigma), 5 mM N-ethylmaleimide (Sigma), and 10 mM NaF and incubated for 2 h on ice and pelleted down. The supernatants were incubated overnight with FLAG M2 Magnetic Beads (Sigma) at 4°C. 3× FLAG tag peptide (Sigma) was used to elute the immunoprecipitated complexes. Two thirds of the eluted complexes were boiled for 5 min with 1% SDS and diluted in 1:10 with lysis buffer. The diluted probes were reimmunoprecipitated with HA Magnetic Beads (Invitrogen) for 2 h at room temperature. The immunoprecipitated complexes were boiled in 2× Laemmli sample buffer (Sigma) at 95°C and processed for immunoblot analysis.