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In an independent approach, we used subcellular fraction- (1 µM) induces the heterodimerization ofFRB and FKBP12, thereby ation followed by the in vitro NLRP3 assay to study the correlation recruiting the phosphatase to the TGN to hydrolyze its target phospholipid. [...] a, HeLa cells stably expressing the indicated proteins were nigericin in the presence of increasing concentrations of KC!, and the treated with nigericin followed by the in vitro NLRP3 activity assay. [...] Mutations of residues in the KKKK motif to ment bound to several negatively charged phospholipids, and the alanine inhibited the ability of full-length NLRP3 to form puncta in a binding was abolished by the 4KA mutation (Extended Data Fig. [...] expressing the indicated NLRP3 proteins were treated with imiquimod in The percentage of cells with NLRP3 puncta on the dTGN was quantified the presence of increasing concentrations of KC! and the cell lysates were from 100 cells (n = 3, mean± s.d., two-sided t-test). [...] Surprisingly, whereas inhibition ofK+ Ptdins4P binding is important for NLRP3 recruitment to the dTGN efflux repressed the recruitment of wild-type NLRP3 to dTGN and and its subsequent activation, which is consistent with the observation blocked its activation, it had no effect on either constitutive TGN local- that Ptdins4P is enriched on the TGN18.