Fig. 1: GD-NT, but not GD-FL, possesses massive K63-linked polyubiquitin.

A Schematic illustration of mouse GSDMD cleavage, the proteases responsible for the specific cleavage, and the constructs created for the investigation of GSDMD ubiquitination. B Schematic illustration depicts the structure of mouse GSDMD. The structure of mouse GD-NT was downloaded from RCSB PDB (PDB code: 6N9N), and PyMOL was used to generate a structure model of GD-FL, GD-NT. C 293 T cells were transfected with Flag-GD-FL and/or caspase-11. 24 hours (h) later, the cells were harvested and subjected to Flag-IP assay. The precipitants and WCL (whole cell lysate) were immunoblotted with the indicated antibodies. D His-Ub was transfected with Flag-GD-FL and/or caspase-11 in 293 T cells. 24 hours later, the cells were harvested and subjected to Ni-NTA Pulldown assay. The precipitants and WCL were immunoblotted with the indicated antibodies. E Similar to (B), except that 293 T cells were transfected with Flag-GD-FL, NT, or CT, respectively. The mark “*” indicated that the band of GD-CT overlapped with the unspecific band from the anti-Flag primary antibody. F 293 T cells were transfected with GST-GD-FL or NT and treated with or without MG132 (10 μM, 6 h). 24 hours later, the cells were harvested and subjected to GST Pulldown assay. The precipitants and WCL were immunoblotted with the indicated antibodies. G Similar to (C), except that His-Ub was transfected with Flag-GD-FL, NT, or CT, respectively. H His-Ub-WT, His-Ub-K63 only, or His-Ub-K48 only was transfected with Flag-GD-NT in 293 T cells. 24 hours later, the cells were harvested and subjected to Ni-NTA Pulldown assay. The precipitants and WCL were immunoblotted with the indicated antibodies. In the construct His-Ub-K63 only and His-Ub-K48 only, all other lysines of ubiquitin were substituted with arginine except for Lys63 and Lys48, respectively. In C, E and F, to keep the integrity of the ubiquitin chain attached to substrate proteins, NEM (10 mM) was added to inhibit the activity of cysteine peptidases. To prevent the influence of GSDMD interactome, protein samples for IP were denatured via heating at 100 °C for 5 minutes to break protein-protein interaction.