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. 2015 Mar 2;112(11):E1210–E1219. doi: 10.1073/pnas.1418335112

Fig. 5.

Fig. 5.

BRUCE and USP8 both promote deubiquitination and formation of BRIT1 foci. (A) Depletion of BRUCE increases BRIT1 ubiquitination. U2OS cells depleted of BRUCE were transfected with FLAG-BRIT1 and Myc-Ub (K48R). BRIT1 ubiquitination in the chromatin-enriched fraction was assayed. (B) BRIT1 (KR)-1 forms DNA damage foci in BRUCE-depleted cells. U2OS cells depleted of BRUCE were transfected with wild-type BRIT1 or BRIT1 (KR)-1 and were immunostained for BRIT1 foci (anti-FLAG) 1 h after IR (5 Gy) exposure. Foci formed (solid circles) and abolished (dashed circle) are shown. (Scale bars, 10 µm.) (C) Depletion of USP8 increases BRIT1 ubiquitination. U2OS cells depleted of USP8 were assayed for BRIT1 ubiquitination following the method in A. (D) BRIT1 (KR)-1 formed DNA damage foci in USP8-depleted U2OS cells following the method in B. (E) The DUB activity of USP8 mediates BRIT1 deubiquitination. Ub conjugates were isolated from the chromatin fraction of U2OS cells transfected with various constructs as indicated above the blots and were immunoblotted for the proteins indicated at the right. (F) USP8 DUB activity is required for the recovery of BRIT1 foci preinhibited by Ub overexpression. U2OS cells cotransfected with the constructs indicated at the left were irradiated and stained for BRTI1 (green), FLAG (red), and Myc (yellow). Foci formed (solid circles) and abolished (dashed circles) are shown (Scale bars, 10 µm.)