Side-by-side comparison of two reported UAF1-binding sites in USP1 using a nuclear relocation assay. A. Schematic representation of USP1 and the deletion mutants used in the analysis. The two reported UAF1 binding sites are highlighted: in orange, the 235–408 segment reported by Villamil et al. ; in green, the 420–520 segment reported by García-Santisteban et al. . The location of USP1 nuclear localization signals (NLSs, in red), and the S313 phosphorylation site is also shown. S313 wild type and S313D phosphomimetic mutants of USP1(235–408) and USP1(del420-520) were used. B. Confocal images showing representative examples of the results using the in vivo nuclear relocation assay. 293T cells were co-transfected with UAF1-mRFP and GFP-USP1 full length (FL), GFP-USP1(420–520), YFP-USP1(235–408), YFP-USP1(235–408)S313D, YFP-USP1(del420-520) and YFP-USP1(del420-520)S313D. Cells were counterstained with Hoechst to show the nuclei (DNA panels). UAF1-mRFP clearly relocates to the nucleus when co-expressed with GFP-USP1 full length and GFP-USP1(420–520), but not with the remaining deletion mutants. C. Graph showing the results of a semiquantitative analysis of the nuclear relocation assay samples. Slides were coded and the nuclear (N), nuclear/cytoplasmic (N/C) or cytoplasmic (C) localization of UAF1-mRFP was determined in at least 100 cells per slide. The results (mean and SEM) of three independent experiments are shown in the graph.