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. 2011 Jul 22;12(9):924–930. doi: 10.1038/embor.2011.140

Figure 1.

Figure 1

USP22 targets FBP1 for deubiquitination. (A) Scheme for identification of substrates with abundant ubiquitination upon USP22 depletion. (B) Depletion of USP22 leads to increased FBP1 ubiquitination in 293T cells. shControl or shUSP22 293T cells were transfected with HA-Ub, ubiquitinated proteins were precipitated and blotted for FBP1. (C) Overexpression of WT but not catalytically dead (C185S) USP22 decreases FBP1 ubiqutination levels in vivo. The 293T cells were transfected with the indicated vectors, ubiquitinated proteins were purified with an anti-HA-resin, resolved by SDS–PAGE and blotted with the indicated antibodies. The asterisk indicates background bands. USP22 coprecipitates with FBP1. FLAG-FBP1 (D) or endogenous FBP1 (E) was purified, and purified fractions were analysed by immunoblot. Et-Br, ethidium bromide; FBP1, far upstream element (FUSE)-binding protein 1; HA, haemagglutinin; IgG, immunoglobulin G; IP, immunoprecipitation; Ni-NTA, nickel-nitriloacetic acid; shRNA, short-hairpin RNA; Ub, ubiquitin; USP22, ubiquitin-specific protease 22; WT, wild type.