Fig. 1. GSDMD directly interacts with TRIM21.
a Heatmap depicts the levels of proteins identified by mass spectrometry that might interact with GSDMD in iBMDMs treated with or without TSZ (10 ng/mL TNF-α, 100 nM Smac, and 10 μM ZVAD) and LPS plus Nig (iBMDMs were primed with 1 μg/mL LPS for 2 h and then stimulated with 20 μM Nig for 30 min). Immunocomplexes of endogenous GSDMD were subjected to SDS-PAGE. Gels were cut based on heavy-chain IgG bands. The sample was analyzed as described previously [49]. b Co-immunoprecipitation (Co-IP) analysis of GSDMD-Flag with HA-TRIM21 or HA-WWP2 in HEK293T cells. Asterisk (*) symbol indicates, nonspecific binding. c Measurements of the binding affinity between GSDMD and TRIM21 or WWP2 (negative control) by ITC method. The upper panel represents the raw data, revealing the calorimetric response peaks in μWatt during successive injections of GSDMD to the TRIM21 sample cell or the WWP2 sample cell. Individual peaks from titrations were integrated and presented in a Wiseman plot. The lower panel is the best fit of the raw data after subtracting the heat of dilution from the appropriate buffer blank. The solid line in the lower panel shows the best fit of the raw data to the one-set of sites binding model. Apparent Kd of GSDMD to TRIM21 was 16.3 ± 1.8 μM. Titrations were carried out at 25 °C in 20 mM Tris-HCl buffer (pH 8.0) containing 150 mM NaCl. d The schematic diagram of human GSDMD and its truncated mutants. e GSDMD-mCherry or its mutants and TRIM21-GFP were co-transfected into HeLa cells. Lysates were immunoprecipitated with anti-GFP antibody and then immunoblotted with anti-mCherry or anti-GFP antibody. Asterisk (*) symbol indicates nonspecific binding. EV empty vector. f HEK293T cells were transfected with ZsYellow-GSDMD-mCherry, Caspase-4-Flag, and TRIM21-GFP as described. The cells were transfected with 1 μg/mL LPS and incubated at 37 °C for 4 h. Anti-GFP agarose beads were used in the following co-IP analysis. g Endogenous GSDMD binds with TRIM21 in mouse primary BMDMs. BMDM cell lysates were immunoprecipitated with an anti-TRIM21 antibody or IgG as control, followed by anti-GSDMD or anti-TRIM21 immunoblotting. All data are representative of three independent experiments.