Figure 2

The catalytic activity of USP45 is essential for its interaction with Spindly. (A) U2OS cells were transiently transfected with the indicated GFP fusion constructs encoding full-length or indicated fragments of USP45 (left-hand side panel). Thirty six hours post-transfection cells were lysed and GFP immunoprecipitated. The immunoprecipitates were resolved on a polyacrylamide gel and stained with Coomassie (right-hand side upper panel) or subjected to immunoblotting with the indicated antibodies (right-hand side lower panel). GFP empty vector was used as a negative control. (B) U2OS cells were transfected with the indicated USP45 constructs GFP tagged, mCherrySpindly-FKBP and Lin11-FRB for 40 hours. Lin11 expression allows for mislocalisation recruitment at the plasma membrane of the FKBP-tagged protein. Rapamycin (4 μM) was administered for 1 hour (at 37 °C) and then cells were visualized under a fluorescence microscope: USP45 construct (green), Spindly (red). Left-hand side panel DMSO treated samples; right-hand side Rapamycin treated samples. 60X magnification. Scale bars: 5 μm. GFP-USP51 was used as negative control. (C) Yeast two-hybrid (Y2H) assays were performed with a GAL4 DNA-binding domain fusion or activation domain for each protein indicated in the table to detect interaction between the indicated proteins. Cells grown in media lacking LEU, TRP and HIS with increasing concentrations of 3-amino, 1,2,4 triazole (to select for bait and prey plasmids). (A–C) All experiments were performed at least three times and a representative example is shown.