Extended Data Fig. 2: UFD2 prefers to synthesize the K48/K29 branched Ub chain.

a, In vitro Ufd2-dependent ubiquitination assays on K29diUb and K29triUb were performed (E1: Uba1, E2: Ubc4). b, In vitro Ufd2-dependent ubiquitination assays were performed on fluorescent K29diUb mutants. K29diUbPro* refers to K29diUb with lysine to arginine mutation at the proximal Lys48 site and labeled with Oregon Green 488 dye (OG488). K29diUbDis* refers to K29diUb with lysine to arginine mutation at the distal Lys48 site and labeled with OG488. K29diUbWT refers to K29diUb without any mutation at Lys48 site and labeled with OG488. The synthetic routes were depicted in Supplementary Fig 8. c, In vitro Ufd2-dependent ubiquitination assays on eight different linkage-type diUbs were performed. d, The purified products of in vitro Ufd2-dependent ubiquitination reactions on K29diUb and K29triUb were treated with the protease LbPro*. The above gels (a-d) are representative of three independent experiments. e, A schematic representation illustrates the mass spectrometry identification workflow for the Ub chain architectures generated by Ufd2 on K29diUb and K29triUb. f, The representative MS/MS spectrum corresponding to the signature peptide derived from K29-K48 branched linkages (aa 29–57), featuring GlyGly modifications at K29 and K48, is presented. g-h, Intact mass spectrometry (MS) analysis of Lbpro* treated in vitro ubiquitination reactions, and quantification of the relative abundance of each ubiquitin species was shown in (g) using K29diUb as substrate and (h) using K29triUb as substrate from n = 2 independent experiments. Each data point is shown as a black dot [data from (g)].