BTK phosphorylates the PYD-NACHT linker. (A) NLRP3 domains (UniProt ID Q96P20). (B) Flag immunoprecipitation (Flag-IP) from HEK293T cells transfected with Flag-tagged NLRP3 truncation and/or Flag–BTK constructs (n = 3). (C) As in B but including ibrutinib (n = 3). (D) Positions of targeted tyrosine residues. (E) Linker region including polybasic motif. (F) As in B but using NLRP3 Y>F point mutants or WT NLRP3, with WT or KD BTK plasmids (n = 4). (G) Quantification of F (n = 4). (H) WES capillary electrophoresis of NLRP3 Flag-IP from HEK293T cells transfected with WT or mutant Flag-NLRP3 and WT or KD BTK (n = 3). (I) Dot blot of BTK assay with 15-mer NLRP3-derived WT or mutant synthetic peptides (n = 3). After BTK removal using anti-His beads, peptide mixtures were directly spotted and stained with a total peptide stain (input) or anti–p-Y. A circle denotes peptides containing the three PBR tyrosines, and a square denotes Y168-containing peptides. (J) As in F but also NLRP3 linker (WT or Y mutated) fused to mCitrine (mCit)-HA (n = 3). (K) Tyrosines (red) highlighted in the model of NLRP3 (blue)–NEK7 (yellow) complex (PDB: 6NPY). (L and M) Close-up view on dimer interface (L) and putative nucleotide binding site (M). G represents combined data (mean + SD) from n biological replicates (each dot represents one replicate). B, C, F, and H–J are representative of n technical replicates. *, P < 0.05 according to one-sample t test (G). IB, immunoblot; Ibru., ibrutinib; RLU, relative light unit.