Extended Data Fig. 4: Characterization of GZMK substrates and Comparison between GZMK and other C3 convertases.

a, A representative Coomassie blue-stained gel showing the purified GZMK and GZMK^S214A. b, Schematic of a fluorescence resonance energy transfer (FRET)-based protease assay for determining GZMK activity. c, Measurements of GZMK activity using the assay in (b). Lysozyme and Trypsin were included as negative and positive controls, respectively. Each symbol denotes a technical replicate (n = 3), and data are presented as mean±S.D. AU, arbitrary unit. d, Representative immunoblots showing cleavage of SET by GZMK. e, Representative immunoblots showing cleavage of tissue DMBT1 by different doses of GZMK, with β-actin as a sample processing control. Data are representative (a-e) of at least two independent experiments. f, Representative Coomassie Blue-stained gel showing the cleavage of serum-purified C3 by GZMK, C3bBb and C4b2a. g, Statistics summarizing the level of C3a converted by GZMK and C3bBb, as normalized to C4b2a. Each symbol denotes one experiment, and lines denote means. h, Representative HPLC trace (left) and commassie blue-stained gels (right) showing the isolation of C3a. Serum-purified C3 was incubated with recombinant human GZMK or C4b2a assembled from serum-purified components. mAU, milli-absorbance unit. i-j, Immunoblots (i) and summary statistics (j) showing the activation of ERK in THP1 cells by C3a from different sources. Data are representative (f, h and i) or pooled (g and j) from three independent experiments. Each symbol denotes one experiment, and lines denote means. P values by two-sided unpaired t tests. Source images for gels and blots are presented in Supplementary Fig. 1.