Fig 1: All Deltex E3s ADP-ribosylate Ub via the RING-DTC domains.(A) Reduced autoradiogram showing Ub ADP-ribosylation reactions with DTX3L and PARP9 variants in the presence of E1, E2 UbcH5B, Mg2+-ATP, Ub, and 32P-NAD+. The reaction scheme is shown above, where E2~Ub was first generated before the addition of E3 and NAD+ to initiate the reaction. (B) Domain organization of the Deltex family of E3s. All members share adjoining C-terminal RD domains. DTX1, DTX2, and DTX4 have tandem WWE domains at the N terminus and C3H2C3-type RING fingers. DTX3 and DTX3L have C3HC4-type RING fingers. DTX3L has a unique N-terminal region that mediates its interaction with PARP9, while DTX1 to DTX4 contain a proline-rich region in domain D. (C) Western blot of in vitro Ub ADP-ribosylation reactions in which E1, E2 UbcH5B, Ub, Mg2+-ATP, or full-length DTX3L (DTX3L-FL) has been omitted as indicated in the presence of biotin-NAD+ and separated by SDS–polyacrylamide gel electrophoresis (PAGE) in reducing conditions. (D) As in (C), but with DTX3L-RD. (E) Western blot of in vitro reactions with E1, E2 UbcH5B, Mg2+-ATP, Ub, biotin-labeled NAD+, and the indicated Deltex protein RING-DTC domains, pCBL, RNF38, or BIRC7 separated by SDS-PAGE in reducing conditions. For (C) to (E), α-Ub is shown in red, and NeutrAvidin DyLight is shown in green.