USP1-mediated PCNA deubiquitination is not abrogated by the S313A mutation. A. Immunoblot analysis of 293T cells co-transfected with Xpress-UAF1 and YFP-vector, GFP-USP1 wild type (WT) or the catalytically inactive GFP-USP1C90S. Cells were either left untreated (−), or were treated (+) with 4 mM hydroxyurea (HU) for 24 h. Using an anti-PCNA antibody, monoubiquitinated PCNA (ubPCNA) is detected as a band migrating slightly above the non-ubiquitinated form (PCNA). A short-exposure time image showing both PCNA and ubPCNA, as well as a cropped image showing only ubPCNA with longer exposure time are shown. The dotted line indicates that a panel is a composite of two images from a single exposure of the same gel. Expression of wild type GFP-USP1 decreased HU-induced PCNA monoubiquitination, whereas expression of GFP-USP1C90S did not. Expression of β-actin was used as a control for equal loading of the protein samples. B. On the left, representative example of immunoblot analysis of 293T cells co-transfected with Xpress-UAF1 and GFP-USP1 wild type, GFP-USP1C90S, GFP-USP1S313A or GFP-USP1S313D, and treated with 4 mM HU for 24 h. The ratio of ubiquitinated to non-ubiquitinated PCNA (ubPCNA/PCNA ratio) was determined by densitometry analysis of the immunoblot bands. The graph on the right shows the results of this analysis. The ubPCNA/PCNA ratio was similar in cells expressing wild type GFP-USP1, GFP-USP1S313A and GFP-USP1S313D, but higher in cells expressing the catalytically inactive GFP-USP1C90S. The data represent the mean and SEM of 7 independent experiments.