FIG 4.

USP7 binds to ORF45 through a consensus USP7-binding motif. (A and B) GST pulldown assays identified an internal region of ORF45 (aa 210 to 237) capable of binding to USP7. Lysates of FLAG-USP7-expressing HEK293T cells were pulled down with purified GST-tagged fragments as indicated, and the eluates were analyzed by Western blotting. -C, negative control. (C) Representation of the ORF45 fragments used in panels A and B and their abilities to bind to USP7. (D) USP7 and antibody 2D4 bind mutually exclusively to a central region of ORF45. USP7 was coexpressed with ORF45 wild type (WT) or ORF45 G224E/S226A in HEK293T cells. The lysates were immunoprecipitated using antibody 8B8 or 2D4, and the immunocomplexes were analyzed by Western blotting. (E) ORF45 colocalizes with and relocalizes USP7. In the top two rows, HeLa cells were transfected with ORF45, left untreated or treated with LMB, and then stained using rabbit anti-USP7 and mouse anti-ORF45 antibodies and visualized by confocal microscopy. Cells lacking ORF45 signal are marked by arrowheads, and cells with ORF45 signal are marked by arrows. In the bottom row, iSLK.BAC16 cells were induced for 60 h by adding doxycycline and butyrate and then stained using rabbit anti-USP7 and mouse anti-ORF45 antibodies and visualized by fluorescence microscopy.