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![[Figure 2]](ai5017fig2mag.jpg)
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Figure 2
Biochemical characterization of exosite peptides in GSDMD-cleavage TR-FRET and caspase-1 activity assays. (a) Schematic of the GSDMD-cleavage TR-FRET assay. Two fluorophores are used for detection and quantitative analysis of FRET signals: europium cryptate conjugated to anti-FLAG M2 antibody serves as the donor with a maximum emission peak at 620 nm and XL665 conjugated to streptavidin serves as the acceptor with a maximum excitation at 615 nm. An intact N-terminally FLAG-tagged and C-terminally biotinylated full-length GSDMD protein results in emission of the acceptor XL665 at 665 nm following excitation by energy transfer from the donor Eu and cleavage by caspase-1 leads to a decrease in the emission of the acceptor at 665 nm. (b) Schematic of the caspase-1 activity assay. A fluorogenic tetrapeptide (Ac-WEHD-AFC) was used for quantitative analysis of caspase-1 activity. The AFC fluorophore is quenched in the intact peptide, whereas cleavage by caspase-1 results in increased free AFC and emission at 510 nm. (c) GSDMD-cleavage TR-FRET assays (blue circles) and caspase-1 assays (red squares) were performed in the presence of increasing concentrations of peptides in three independent experiments (n = 3). Selective inhibition of GSDMD cleavage compared with nonspecific inhibition of caspase-1 is indicated. Abbreviations: Eu, europium cryptate; SA, streptavidin; B, biotin |
![[Figure 2]](ai5017fig2.jpg)
 | STRUCTURAL BIOLOGY |
ISSN: 2059-7983
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