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

Figure 5.

Figure 5

In Cell Extracts, OTULIN ABP Labels OTULIN and USP5, and Induces PolyUb Chain Formation

(A) Extracts of Flag-OTULIN WT or C129A overexpressing HEK293 cells (∼2.5 × 105 cells/reaction) were incubated with 1 μg OTULIN ABP (30 min, 30°C). Labeling of exo- and endogenous OTULIN was analyzed by western blot.

(B) Cross-reactivity of OTULIN ABP with endogenous DUBs. Incubation of OTULIN ABP in HEK293 extracts was performed as in (A). See also Figure S3A.

(C and D) Extracts of Jurkat T cells (2 × 107 cells) were incubated at room temperature with increasing OTULIN ABP amounts for 15 min (C) or with 1 μg OTULIN ABP for different times (D).

(E) For ATP depletion, extracts from 6 × 105 HEK293 cells were incubated with apyrase prior to the incubation with 1 μg OTULIN ABP for 30 min. Ubiquitin chain formation and ABP adducts were analyzed by western blot. See also Figure S3B.