Fig. 4

Cleavage assays of purified Et-OTU on di-Ub WT substrates in vitro. a Purified Et-OTU DUB was incubated with the K48-, K63-, K11-, K29-, K33-, or K6-linked di-Ub chains for 30 min and resolved by Western blotting. b Purified Et-OTU DUB was incubated individually with the K48- and K6-linked di-Ub chains for the indicated times. The hydrolytic efficiency of Et-OTU DUB towards K6-linked di-Ub chains was higher than that of Et-OTU DUB towards K48-linked di-Ub chains. c Catalytic residues (Cys residue 239, Asp residue 236 and His residue 351) of Et-OTU were individually mutated to Ala. Purified OTUD236A, OTUC239A, OTUH351A and WT OTU DUBs were individually incubated with K48 and K6 di-Ub chains for 30 min