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. 2009 Feb 20;284(8):5343–5351. doi: 10.1074/jbc.M808430200

FIGURE 1.

FIGURE 1.

Purification of native UAF1 complexes containing USP1 and other deubiquitinating enzymes. A, Flag-HA-tagged UAF1 was immunoprecipitated from either HeLa cell extracts or cell extracts prepared from HeLa cells where the endogenous USP1 protein is stably knocked down by shRNA. The immunoprecipitate was analyzed by immunoblotting using the indicated antibodies. B, the immunoprecipitates from A were analyzed for associated deubiquitinating enzyme activity using ubiquitin-AMC as a substrate. C, silver stain of the UAF1 complexes. D, glycerol gradient sedimentation of the UAF1 complexes. Fractions were separated by SDS-PAGE, and proteins were visualized by silver stain. Subcomplexes as determined by sedimentation profile are indicated by roman numerals I-IV. Sedimentation profile of molecular weight markers is indicated. E, the fractions from C were analyzed by immunoblotting using the indicated antibodies (upper panels) and for deubiquitinating enzyme activity using ubiquitin-AMC (lower panel).