Extended Data Fig. 9: Validation of Ufd2 interactions with K29triUb and exploration of Ufd2 dimerization.

a, Structure model of triUb monomeric complex. b, In vitro Ufd2-dependent ubiquitination assays using fluorescent labeled K29triUb as substrate. Various Ufd2 mutations, including L347G, F361G, Y204R, D365K, D369K, D299K, H300G, D299K&H300G, H576G, and D360A, were analyzed. The gel image is representative of independent biological replicates (n = 3). c, Bar graph comparing the fraction of reacted K29triUb and show the mean ± SD from n = 3 independent experiments. Each data point is shown as a black dot [data from (b)]. A two-sample, two-tailed Student’s unpaired t test was employed to calculate p values; ****p < 0.0001. d, A representative size-exclusion chromatography with multi-angle light scattering (SEC-MALS) of Ufd2, with peaks corresponding to the Ufd2 dimer and monomer labeled. e, Surface Plasmon Resonance (SPR) measurement of binding of wild-type Ufd2 to wild-type Ufd2 or Ufd2 dimer mutant lacking the dimeric helix (residue 419-439). f, Bis(sulfosuccinimidyl) suberate (BS3) crosslinking analysis of wild-type Ufd2 and Ufd2 dimer mutant. These gels are representative of two independent experiments.g, In vitro Ufd2-dependent ubiquitination assays were performed to compare the ubiquitination efficiency of Ubc4 wild-type and Ubc4 S23R, utilizing fluorescently labeled K29-triUb as the substrate. Gel images are representative of independent biological replicates (n = 3). h, The line graph illustrates the proportions of K29-triUb in the reactions, and show the mean ± SD from n = 3 independent experiments.