Pre-cleared cell lysates were used for the co-immunoprecipitation (Co-IP) analysis using a Pierce Co-IP kit (Thermo Scientific Pierce) and following the manufacturer’s instructions. This kit provides covalent antibody immobilization onto an insoluble agarose support, and then incubated with a cell lysate containing the target protein. We use the purify mouse anti-NLRP3 antibody (10 μg, AdipoGEn) to immunoprecipitate the antigen (bait protein, IB) which bind the interacting protein (prey protein, IP) caspase-1 (1/100, Santa Cruz). Upon elution the immune complexes, the samples were resolved with 15% SDS-PAGE and transferred to PVDF membranes (Bio-Rad) for immunoblotting. The mouse IgG was used as a negative control.
