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. 2020 May 11;11:2343. doi: 10.1038/s41467-020-15985-4

Fig. 6. The UBD domain is critical for optimal cleavage of K48 and K63 di-ubiquitin substrates.

Fig. 6

a WT or V203D OtDUB1–259 was preincubated with an equimolar concentration of ubiquitin then diluted to 0.5 with 1 µM K63 or K48 di-ubiquitin. Samples were resolved by SDS-PAGE and SYPRO Ruby stained. Source data are provided as a Source Data file. b Quantification of K48 di-ubiquitin cleavage from a with the mean of three experiments and SD bars are shown. Source data are provided as a Source Data file. c WT or E238A/R242A OtDUB1–259 was preincubated with an equimolar concentration of ubiquitin and then diluted to 0.5 with 1 µM K63 or K48 di-ubiquitin. Samples were processed as in a. Source data are provided as a Source Data file. d Quantification of K48 di-ubiquitin cleavage from c with the mean of three experiments and SD bars are shown. Source data are provided as a Source Data file. e WT or F59T OtDUB1–259 was preincubated with an equimolar concentration of ubiquitin and then diluted to 0.5 with 1 µM K63 or K48 di-ubiquitin. Samples were processed as in a. Source data are provided as a Source Data file. f Ub-AMC-cleavage assay comparing the activities of OtDUB1–177, WT, and V203D OtDUB1–259. Ub-AMC (400 nM) was incubated alone (Blank) or with the indicated OtDUB (350 pM). Lines represent the average of a technical triplicate with SD bars at each 40 s time point. g Quantification of K63 di-ubiquitin cleavage from e with the mean of three experiments and SD bars are shown. Source data are provided as a Source Data file. h OtDUB1–177, OtDUB1–259, and OtDUB1–259-V203D were preincubated with an equimolar concentration of ubiquitin before adding to 1 µM of K63 tetra-ubiquitin chains to obtain 0.5 µM of enzyme. Samples were processed as in a (n = 2). Red asterisks indicate enzyme protein bands. Source data are provided as a Source Data file. Unpaired, two-tailed t tests were performed (b, d, g) for pair-wise comparisons between OtDUB1–259 WT and mutant variants for each condition and time point (*p < 0.05, **p < 0.005, ***p < 0.0005, n.s. = not significant).