ASC SUMOylation was analysed in HEK-293T cells by enriching His-SUMO using nickel-nitrilotriacetic acid (Ni-NTA) coupled agarose beads or enriching HA-ASC with HA Nanoab Mag Beads as previously described33. Endogenous SUMOylated ASC were enriched by immunoprecipitations following the published protocol63,64 with minor changes. Briefly, 5 × 107 of cells were lysed in 1 mL of lysis buffer (20 mM Phosphate buffer pH 7.4, 150 mM NaCl, 1% Triton, 0.5% Na-deoxycholate, 1% SDS, 5 mM EDTA, 5 mM EGTA, 1 μg/mL aprotinin, 1 μg/mL leupeptin, 1 μg/mL Pepstatin A, 0.1 mM Pefabloc, 20 mM N-ethylmaleimide (NEM), protease and phosphatase inhibitors (Sigma-Aldrich)). The lysate was sonicated to reduce its viscosity, then diluted 1:10 with RIPA buffer without SDS and incubated with antibody-coupled beads overnight at 4 °C. Beads were washed three times with high-salt buffer and then boiled for SDS-PAGE immunoblotting analysis.
