Fig. 4. TRIAD3 selectively ligates unanchored, K63-linked ubiquitin chains in vitro.

a Scheme of ubiquitin mutants used in (4b), illustrating ubiquitin’s seven internal lysines (K) and their replacement by arginine (R), shown in red. b GST-fused TRIAD3-RBR⁸⁴⁵ fragment was incubated with wt ubiquitin or different ubiquitin mutants as illustrated in a. As negative controls, reactions were incubated without ATP or with GST instead of GST-TRIAD3-RBR⁸⁴⁵. Samples were analysed by western blotting and ubiquitin chains were detected using anti-ubiquitin antibodies (upper panel), and GST-TRIAD3-RBR fragments were detected using anti-GST antibodies (lower panel). * denotes multi-mono-ubiquitylated GST-TRIAD3-RBR in K11, K48, and K0 ubiquitin samples. c Ubiquitin chains synthesised by recombinant TRIAD3A were analysed by UbiCRest with K48-specific (OTUB1) or K63-specific (AMSH) de-ubiquitylating enzymes or GST as control. Ubiquitin chains and GST and GST-fused de-ubiquitylating enzymes were detected by immunoblotting with anti-ubiquitin (upper panel) and anti-GST antibodies (lower panel), respectively