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

Figure 2.

Figure 2

Recombinant OTULIN Is Labeled by the OTULIN ABP

(A) Recombinant OTULINcat (1 μg) was incubated with 4 μg bio-UbG76Dha-Ub (2 hr, 37°C). OTULIN-diUb complex formation was analyzed by Coomassie staining.

(B) OTULINcat (250 ng) was incubated at 30°C in a 1:1 molar ratio with OTULIN ABP. OTULIN-diUb complex formation was monitored by Silver staining.

(C) OTULINcat (250 ng) was incubated with increasing OTULIN ABP concentrations (15 min, 30°C). OTULIN-diUb complex formation was monitored by Silver staining.

(D) TetraUb cleavage assay was performed after pre-treatment of OTULIN with increasing ABP concentrations. TetraUb chain cleavage was visualized by western blot.

(E) Left: TetraUb cleavage assay was performed as in (D) in the presence of increasing PR-619 concentrations. Right: OTULINcat (500 ng) was pre-treated with PR-619 before incubation with 2 μg OTULIN ABP (5 min, 30°C). OTULIN-diUb complex formation was analyzed by Silver staining.