Fig 1: GSDMD-BS activity is dependent on NTD-CTD intramolecular interaction. (A) Luciferase assay of GSDMD-WT and GSDMD-MUT. Upper panel: Luciferase assays. GSDMD-BS activity was confirmed by introducing several mutations in the CTD responsible for binding the repressing the NTD. GSDMD-MUT had a considerably low signal compared to GSDMD-BS, demonstrating GSDMD-BS signal is attributed to luciferase complementation upon NTD-CTD binding (mean ± SD; n = 3). Lower panel: Equivalent amounts of lysates (Mock, GSDMD-WT, GSDMD-MUT) used for luciferase assays (upper panel) were also used for Western blot analysis using anti-Nluc antibody. (B) Reduced GSDMD-BS activity after CASP1 cleavage of GSDMD. GSDMD-BS was transfected alone or together with CASP1-FLAG into HEK293T cells, then protein lysate was extracted for luciferase assay and Western blot. Upper panel: Luciferase assays. In the presence of catalytically active CASP1, the GSDMD-BS luminescent signal drops dramatically (~90 000-fold; *, p < 0.05) compared to GSDMD-BS alone (mean ± SD; n = 2). Lower panel: Western blot analysis of GSDMD-BS and CASP1-FLAG levels by Western blot using anti-GSDMD (detect GSDMD-NTD) and anti-FLAG (detecting CASP1-FLAG). While full-length GSDMD-BS (75 kDa) is present when expressed alone, cleaved GSDMD (51 kDa; cleaved component contains GSDMD-NTD and LgBiT) is only detected when co-transfected with CASP1.
Fig 2: Characterizing GSDMD-BS activity in vitro. (A) Purification of His-GSDMD-BS fusion protein. The His-GSDMD-BS was purified and quantified using BSA as a standard (0.25–4 μg). Fusion protein is estimated to be approximately 0.25 μg/μL from the Coomassie blue staining. (B) Luciferase assay of His-GSDMD-BS in vitro. Increasing amounts (0–80 ng) of purified fusion protein were use for luciferase assays in vitro, demonstrating the biosensor’s high sensitivity at relatively low quantities (mean ± SD; n = 2), along with a steady increase in protein levels from the Western blot. *, p < 0.05; **, p < 0.01. (C) Caspase-mediated cleavage of GSDMD-BS in vitro. Purified CASP1 cleaves His-GSDMD-BS fusion protein (100 ng) at a high rate. Upper panel: Luminescent signal reduces drastically when His-GSDMD-BS is incubated with increasing amounts [0–1 Unit (U)] of CASP1 (mean ± SD; n = 2; **, p < 0.01; ***, p > 0.001). Lower panel: Increased cleaved GSDMD (50 kDa) is detected using anti-GSDMD antibody, along with a steady increase in CASP1 levels detected using anti-CASP1 antibody.
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