Extended Data Fig. 10: Characterization and mapping of the pro-caspase-11 and NLRP3 interaction.
From: Caspase-11 interaction with NLRP3 potentiates the noncanonical activation of the NLRP3 inflammasome
a, Schematic indicating the different truncated mutants of NLRP3 used for co-immunoprecipitation experiments in 293 T cells. All forms were fused to 3xHA tag in N-Terminus. b, Schematic indicating the different truncated mutants of casp11C254A used for co-immunoprecipitation experiments in 293 T cells. All forms were fused to 3xFLAG tag in N-Terminus. c, Immunoprecipitation and immunoblotting for coimmunoprecipitating and WCE proteins of overexpressed FLAG-caspase-11 either casp11C254A FL, Casp-11 C254A ΔCARD or Casp-11 C254A ΔSCAF with or without HA-NLRP3FL in 293 T cells 24 h post-transfection. To equilibrate the levels of FLAG-caspase-11 mutants in the immunoprecipitates, 4 times more protein extracts were submitted to anti-FLAG immunoprecipitation when FLAG-casp11C254A ΔSCAF was expressed (labelled 4X) compared to when either FLAG-casp11C254A FL or FLAG-casp11C254A ΔCARD were expressed (labelled 1X). Therefore, note that all proteins from FLAG-casp11C254A ΔSCAF samples (including HA-NLRP3FL) were 4 times more abundant during anti-FLAG immunoprecipitation, yet HA-NLRP3FL was still much less co-immunoprecipitated with FLAG-casp11C254A ΔSCAF compared to FLAG-casp11C254A FL. Immunoblotted proteins are indicated to left of each immunoblot panel. d, Schematic representation of CARD, DED, CASPASE p20 and CASPASE p10 domains of murine inflammatory and apoptotic caspases. SCAF domain is composed of CASPASE p20 and CASPASE p10 domains. All caspases, with the exception of the short forms of mouse and human caspase-12, have SCAF domains. The alignment E values were calculated using the NCBI alignment tool. e, Similarity coefficients between caspase-11 and other murine caspases. f, Protein sequence alignment for murine caspases. The alignment diagram was generated using the alignment module of SnapGene software. g, Schematic indicating the different caspase-11C254A and caspase-9 chimeras used for co-immunoprecipitation experiments in 293 T cells. All forms were fused to 3xFLAG tag in N-terminus.
