Figure 2.

Discovery of Tyrosine as a Target Residue for ADPr

(A) Autoradiogram showing ADPr of H3 peptide (1–20aa) with Ser10 substituted by Ala, Thr, Tyr, Glu, and Asp, alongside Lys9 substituted by Arg and Ala. Coomassie staining of the SDS-PAGE is included.

(B) High-resolution ETD fragmentation spectrum of an HPF1 peptide modified by ADP-ribose on tyrosine 238. The chemical structure of ADP-ribose is depicted (see also Figure S2B). ^∗1, peaks corresponding to co-isolated species in their original charge state. Multiple species in charge states 2–5 passed through the quadrupole and could not be completely deconvoluted.

(C) High-resolution ETD fragmentation spectrum of an HPF1 peptide modified by ADP-ribose on serine 97. The chemical structure of ADP-ribose is depicted.

(D) Autoradiogram showing a panel of PARPs incubated with HPF1 protein. Reaction with mono(ADP-ribosyl)ating PARP1 E988Q (EQ) mutant enhances detection of the HPF1 ADPr. Coomassie staining of the SDS-PAGE is included.

(E) Autoradiogram showing PARP1 E988Q-mediated ADPr of HPF1 WT, HPF1 R239A, and GST-HPF1 proteins. Coomassie staining of the SDS-PAGE is included.

(F) 293T cells were transfected with the same amount of EV or plasmid expressing WT, S97A, or Y238A FLAG-tagged HPF1 protein and left untreated or treated for 10 or 120 min with H₂O₂. Inputs and FLAG-IPs were analyzed by western blotting. CMV, cytomegalovirus.

(G) 293T parental or PARP1 KO cells were transfected with the same amount of EV or plasmid expressing WT FLAG-tagged HPF1 protein and left untreated or treated for 10 or 120 min with H₂O₂. Inputs and FLAG-IPs were analyzed by western blotting.