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. 2017 Oct 19;24(10):1299–1313.e7. doi: 10.1016/j.chembiol.2017.08.006

Figure 4.

Figure 4

OTULIN ABP Shows Minimal Cross-Reactivity with Other Recombinant DUBs

(A and B) DUBs (0.5–1 μM) were incubated with 3 μM OTULIN ABP (1 hr, 30°C). Labeling was analyzed by Silver staining. Asterisks: DUB-diUb adducts. See also Figures S2A and S2B.

(C) Close-up of the structure of the catalytic site of OTULINcat (aa 80–352; white surface) reacted to the bio-UbG76Dha-Ub probe (green cartoon). Insert, OTULIN residues that form the substrate channel are shown in stick representation.

(D) Close-up of structure of the catalytic site of zebrafish CYLD (zCYLD) (aa 578–951, C598S; white surface) bound to Met1-linked diUb (green cartoon) (PDB: 3WXE). Insert, residues from CYLD that form a tight substrate channel around the Gly76-Met1 peptide bond are shown in stick representation.

(E) Recombinant OTULINcat, USP21cat, or CYLD (1 μg) were incubated with either M1-linked diUb WT or UbG76A-Ub (1 hr, 37°C). diUb cleavage was analyzed by western blot.

(F) ST-OTULINcat (WT or C129A) was pulled down with Strep-Tactin beads in the presence of M1-linked diUb or UbG76A-Ub. Interaction between diUb and OTULINcat proteins was analyzed by western blot.