Figure 1. Expression and activity of the Dtx3L/Parp9 heterodimer.

(A) Box and whisker plots showing expression of Dtx3L and Parp9 in cancer (blue) compared to adjacent normal tissue (yellow) for prostate and breast cancer, and nine additional cancers (Figure S1). RNA-Seq by Expectation-Maximization (RSEM) values from TCGA were used to plot normalized expression, whiskers represent the minimum and maximum data values, and significance was calculated using a paired t-test.

(B) Recombinant Dtx3L/Parp9 cleaves NAD⁺. Ubiquitylation reactions containing ³²P-NAD⁺ were analyzed by TLC and autoradiography, with unlabeled ADP-ribose as a migration standard.

(C) Dtx3L/Parp9 ADP-ribosylates Ub. Ubiquitylation reactions with purified Dtx3L/Parp9, histones, and chromatin in the presence of ³²P-NAD⁺. The reactions were subjected to SDS-PAGE, Coomassie Blue (CB) staining (Figure S3D), and autoradiography.

(D) Ubiquitylation reactions with different combinations of human Dtx3L and Parp9 purified from insect cells. Biotin-labeled NAD⁺ was added to monitor ADP-ribosylation of Ub (FL-Neutra detection).