Fig 2: Identification of USP7 region required for interaction with vIRF-2. (A) Transfection-based coprecipitation assays to assess interaction of Flag-tagged vIRF-2 (v2) and positive-control vIRF-1 (v1) and vIRF-3 (v3) proteins, with CBD-fused USP7 NTD (U7NTD) and full-length USP7 (U7, positive control; isoform 2; NP_001273386.1). The identities of USP7 bands on the CBD blots and vIRF bands on the Flag blots are indicated (arrowheads). The dotted line indicates a deleted (irrelevant) region of the CBD blot. (B) Coprecipitation analysis of vIRF-2 interactions with individual and combined domains of USP7, derived from bacteria as His6-fusion proteins, and CBD fusions of vIRF-2 or vIRF-4 (positive control for USP7 NTD binding) expressed in transfected cell extracts. Chitin bead precipitates were His6 immunoblotted for detection of coprecipitated USP7 derivatives. (Right) Input USP7-His6 proteins were validated and compared by Ponceau staining. (C) Coprecipitation analysis using eukaryotically expressed, CBD-fused USP7 fragments comprising residues 1 to 220 (A) and 1 to 207 (NTD) (B) (USP7 sequence NP_001273386.1) and bacterially expressed, purified GST fusions of vIRF-2 residues 226 to 275 (1) and 241 to 260 (2) (each containing USP7-binding sequences, as shown in Fig. 2B). The CBD-fused USP7 fragments were affinity precipitated (AP) with chitin beads, and coprecipitated GST-fused vIRF-2 peptides were identified by GST immunoblotting. Empty vector (vec) transfectant extract provided a negative control. Major full-length protein species (appropriately migrating; size markers not shown) are indicated by arrowheads; higher and lower bands likely represent posttranslationally modified and degradation products, respectively. The dotted line in the precipitation blots indicates repositioning of lanes (from the same membrane). (D) CBD-fused residues 1 to 220 of USP7 (WT; isoform 2; NP_001273386.1) or triplet alanine substitutions thereof (m1 to m4) were used in chitin bead affinity precipitations along with Flag-tagged vIRF-2, each protein pair being derived from transfected HEK293T cell lysates. Empty vector (vec) provided a negative control. (E) In vitro coprecipitation assay employing bacterially expressed and purified GST-fused vIRF-2 residues 241 to 260, or GST (negative control), and His6-tagged USP7 NTD (USP7 residues 1 to 206), USP7 residues 1 to 220, or the m1 variant of the latter (His6-1-220m1; see panel D). Dotted lines indicate deleted (irrelevant) lanes. Precip., precipitation.

Fig 3: The Ubl2 domain of USP7 is required for interaction with p65. A, schematic representation of USP7 mutations used to test interaction with p65. USP7 contains an N-terminal MATH domain, a central catalytic domain (CD), and five C-terminal Ubl domains. B–F, HEK293T cells were transfected with p65 and the indicated FLAG-tagged USP7 plasmids. USP7 was immunoprecipitated (IP) from whole cell lysates using anti-FLAG antibody and analyzed by Western blotting (WB) with anti-p65 antibody. The expression of p65 and USP7 in lysates used for immunoprecipitation (input) was measured by Western blotting using anti-FLAG and anti-p65 antibodies. Western blotting of inputs with anti–ß-actin was used to verify equal protein loading. The positions of molecular mass markers are indicated to the right of each Western blot. The data are representative of at least three independent experiments.

Fig 4: Effect of DNA damage on Cry1-Hausp interaction in transfected 293T cells.HAUSP-V5, FLAG-Cry1, and Actin were detected by IB in IPs or whole cell lysates (WCL) from 293T cells transfected with the indicated plasmids (by calcium phosphate method) and treated with doxorubicin.DOI: http://dx.doi.org/10.7554/eLife.04883.011

Fig 5: Quantitation of in vivo Cry1 ubiquitination.Quantitation of ubiquitinated Cry1 immunoprecipitated from MEFs expressing shRNA targeting Hausp (blue) or a control sequence (black). Left, western blot from Figure 2C with boxes used for quantitation. The average signal detected in the first two lanes (background nonspecific signal from Cry1-/-;Cry2-/- cells) was subtracted from each of the other lanes to generate the data show on the right.DOI: http://dx.doi.org/10.7554/eLife.04883.009