Extended Data Fig. 8: Analysis and verification of the identified proteolytic substrates upon caspase-3 activation.

a, Schematic of the workflow for CAGE-prox-enabled temporal profiling of the proteolytic substrates immediately after caspase-3 activation. b, Venn diagram showing a summary of numbers of the proteolytic substrates identified from three independent temporal profiling experiments. A total of 544 proteins was commonly identified from all the three experiments. c–e, Sequence analysis of the cleavage sites in the identified proteolytic substrates. The logos were generated using Icelogo (https://iomics.ugent.be/icelogoserver/), with all the identified cleavage sequences aligned (the cleavage site is between the P1 and P1′ position). The sequence logos of cleavage sites at all 20 amino acids (526), Asp only (79) and Glu only (29) are shown in c, d and e, respectively. Although Asp is the most abundant cleavage site, the background of other non-caspase sites is relatively high in our results, owing to the lack of N terminus enrichment. The typical DEVD/E motifs were not observed directly, but the amino acid pattern at the P1′ position is similar to that found in previous studies⁴¹. f, Venn diagram comparing the substrates identified from this work with the proteolytic sites recorded in DegraBase (https://wellslab.ucsf.edu/degrabase/). A total of 773 overlapping substrates was found, including 544 substrates that were identified with Asp-containing peptides. g, The expression level of ONBY-incorporated caspase-3 (M61–ONBY) and the corresponding endogenous caspase-3. n = 2. h, Verification of the in vitro cleavage assay using actin, caspase-3 and PARP as positive controls. n = 2. i, Verification of the cleaved and secreted HMGB1 in the culture medium. HMGB1–Flag and wild-type caspase-3 were co-transfected into HEK293T cells, and the cell culture medium were concentrated before immunoblotting analysis. n = 2. j, Alanine screening mapped the caspase-3’s cleavage site on HMGB1 as D139/D140. n = 2. k–v, Verification of the newly identified proteolytic substrates of caspase-3. Recombinant caspase-3 protein was added into cell lysate of HEK293T followed by immunoblotting analysis. n = 2. w, Comparison of the cleavage kinetics of ATP6V1A versus ATP6V1B by caspase-3. After the recombinant wild-type caspase-3 was added into the cell lysate, the mixture was incubated at 37 °C for 0, 0.5, 1, 2, 3, 4 and 6 h, followed by immunoblotting analysis. n = 2. All above-mentioned samples are biological replicates.