Extended Data Fig. 2: Identification of MLKL PARylation sites by PARP1.

a, Schema illustrating mono(ADP-ribosyl)ated (MARylated) MLKL with mass spectrometry. Recombinant MLKL and recombinant PARP1 were incubated together in cell free reaction buffer and treated with poly(ADP-ribose) glycohydrolase (PARG) to acquire MARylated MLKL. MARylated MLKL was analyzed with mass spectrometry to identify amino acid residue that is modified by ADP-ribose. b, Identification of MLKL PARylation sites. MARylated MLKL was isolated with SDS-PAGE. The gel slices containing desired proteins were analyzed with mass spectrometry to identify amino acid residue that is modified by ADP-ribose. c, Schema illustrating the construction of MLKL^WT ECs, MLKL^E351A ECs, MLKL^T253A ECs or MLKL^E351A/T253A ECs (HUVECs). shRNA targeting the intron of MLKL mRNA was introduced into HUVECs to construct MLKL-null ECs. MLKL^WT, MLKL^E351A or MLKL^E351A/T253A double mutants were introduced into MLKL-null ECs via lentivirus to construct MLKL^WT ECs, MLKL^E351A ECs, MLKL^T253A ECs or MLKL^E351A/T253A ECs. d, MLKL protein structure was analyzed with AlphaFold2. It is predicted that modification on MLKL E351 and T253 alter MLKL protein conformation to facilitate MLKL phosphorylation. e, f, MLKL^WT ECs or MLKL^E351A/T253A ECs (HUVECs) were treated with or without 5 μg/mL CBP for 48 hours. The level of cell death was determined by PI staining and analyzed with flow cytometry (e). Quantification of percentage of PI⁺ cells was shown (f) (n = 5 biologically replicates). ****P < 0.0001 was calculated using two-way ANOVA followed by Tukey’s test as post-hoc analysis for f.

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