UAF1 binds and stabilizes a non-phosphorylatable S313A mutant version of USP1 in cell-based assays. A. Confocal images show representative examples of 293T cells co-expressing UAF1-mRFP with YFP (vector), GFP-USP1 wild type (WT), GFP-USP1S313A or GFP-USP1S313D. Left panels show live cell images, whereas right panels show images of fixed cells. Fixed cells were counterstained with Hoechst to show the nuclei (DNA panels). UAF1-mRFP is cytoplasmic when co-expressed with YFP, but relocates to the nucleus when co-expressed with GFP-USP1 wild type, GFP-USP1S313A and GFP-USP1S313D. B. Co-IP analysis of co-transfected 293T cells, showing that Xpress-UAF1 readily co-immunoprecipitates with GFP-USP1 wild type, GFP-USP1S313A and GFP-USP1S313D. A section of the membrane stained with Ponceau is shown to gauge protein loading. WCE, whole cell extract. C. Immunoblot analysis of 293T cells transfected with GFP-USP1 wild type, GFP-USP1S313A and GFP-USP1S313D, alone (−) or in combination with Xpress-UAF1 (+). Anti-GFP antibody was used to detect GFP-USP1 proteins and anti-Xpress antibody was used to detect Xpress-UAF1. β-actin was used as a loading control. UAF1 co-expression markedly increases the levels of GFP-USP1 wild type, GFP-USP1S313A and GFP-USP1S313D to a similar extent. The lower molecular weight band in (+) samples corresponds to the well-characterized N-terminal fragment that results from USP1 autocleavage at the G670/G671 diglycine motif (see below).