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. 2012 Oct 26;287(51):43007–43018. doi: 10.1074/jbc.M112.386938

FIGURE 3.

FIGURE 3.

Co-expression with catalytically inactive DUBs promotes the accumulation of ubiquitylated BRAP. HEK293T cells were transfected with FLAG-ubiquitin, GFP, or GFP-tagged active or inactive (CS) mutants of USP4 and -15 together with empty vector (vector) or myc-tagged BRAP or a mutant BRAP that is unable to recruit a ubiquitin conjugating E2 (WA). Cells were processed as described in Fig. 2, and immunoprecipitates (IP) were probed with anti-FLAG and anti-myc antibodies (A) or anti-GFP antibodies (B) to assess the ubiquitylation status of both BRAP (A) and USP4 and 15 (B). A, pink arrows indicate higher molecular weight bands reactive to anti-myc (BRAP) and anti-FLAG (Ub) antibodies in cells co-expressing wild-type BRAP (wtBRAP) and catalytically inactive USP4 (U4 CS) and USP15 (U15 CS). B, catalytically inactive USP15 and to a lesser extent also USP4 present with higher molecular weight bands that can be detected with FLAG-ubiquitin (blue arrows; see also Fig. 3). U4, USP4; U15, USP15; CS, catalytically inactive mutants; WA, E2-binding deficient mutant. Lane 7, Mr marker. IB, immunoblot.