Fig 4.

Human USP22 requires complex formation for deubiquitinating activity. (A) (Top) Purified recombinant human USP22 or catalytically inactive USP22-C185S was incubated with either DMSO or 5 μM UbVS. A representative gel stained with Coomassie brilliant blue is shown. (Middle) Reactions were performed as described above with various concentrations of DTT. (Bottom) Kinetic deubiquitinating activity assay of recombinant USP22 or the catalytic core of USP2 (USP2cc) with the substrate Ub-AMC. Data are presented in relative fluorescence units (RFU). (B) Purified intact mononucleosomes were incubated with full-length or catalytic inactive (CS) recombinant human Dubs and immunoblotted with the indicated antibodies. (C) Equal amounts of whole-cell lysates from HEK 293T cells were treated with DMSO or 5 μM UbVS for the indicated times and immunoblotted with the indicated antibodies. (D) (Left) Whole-cell lysates from FLAG-HA-USP22-expressing HEK 293T cells were separated by gel filtration and immunoblotted; (right) fractions from gel filtration samples were pooled on the basis of size, purified by IP, and treated with DMSO or 5 μM UbVS. FPLC, fast-performance liquid chromatography.