TRIAD3/RNF216 E3 ligase specifically synthesises K63-linked ubiquitin chains and is inactivated by mutations associated with Gordon Holmes syndrome

a Schematic representation of the C-terminal part of TRIAD3B showing RING1 (R1), In-between-RING (IBR), and RING2 (R2) domain (RBR domain) and proline-rich region (Pro). Disease mutations are depicted in red. Mutation of the ubiquitin acceptor site in RING2 is depicted in grey (C745A). b Overexpressed wt and mutant V5-tagged TRIAD3B proteins were immunopurified from HEK293T cell lysates and incubated in in vitro-ubiquitylation reactions. Free ubiquitin chains were detected by immunoblotting with anti-ubiquitin antibody (top panel). Immunopurified TRIAD3B was detected by western blotting using anti-V5 antibody (*, auto-ubiquitylated TRIAD3B