Extended Data Fig. 9 |. VbP displaces NLRP1 from DPP9 in vitro and in cells.

a, Schematic of the on-bead displacement experiment. The DPP9-ternary complex is expressed in 293T cells, which are then lysed and incubated with FLAG beads. Once bound, beads are split equally and washed with compounds or DMSO. The remainder of the protein is eluted off of the beads. MeBS, Bestatin methyl ester. b, Two structurally distinct VbP inhibitors, VbP and 8J, displace DPP9 from NLRP1-S1213A by the on-bead displacement assay. Anti-FLAG (S1213A-FL), anti-MYC (NLRP1-CT), and anti-V5 (DPP9) antibodies were used in the immunoblots. Representative of 2 independent experiments. c, Schematic of the dTAG experiment. FKBP12 with the F36V mutation (dTAG) is fused to the N-terminus of NLRP1-FL. The dTAG13 ligand recruits an E3-ligase to FKBP12-F36V, leading to its ubiquitination and N-terminal degradation of the fusion protein. NLRP1-CTs (UPA-CARDs) resulting from FIIND autoprocessing are released to assemble the inflammasome. d, NLRP1 FIIND-SA expression in reconstituted HEK293T inflammasome system rescues GSDMD cleavage resulting from dTAG13-induced NLRP1 degradation. VbP prevents GSDMD rescue without inducing additional NLRP1 degradation. Anti-HA (dTAG-NLRP1-FL, dTAG-NLRP1-NT), anti-FLAG (FIIND-SA), anti-GSDMD, and anti-GAPDH antibodies were used in the immunoblots. p30: GSDMD N-terminal fragment from caspase-1 cleavage. Representative of 2 independent experiments.