Ube1 phosphorylation and impact on NER in quiescent B lymphocytes. (A) Representative immunoblot showing the amount of ubiquitin-activating enzyme Ube1 in 50 μg of total cell lysate from wild-type (WT) or XP-E lymphoblasts, and from proliferating (Bp) or quiescent (Bq) B lymphocytes (n = 20 donors). Western blotting was performed with an antibody directed against both the E1A (full length, phosphorylated) and E1B (truncated) isoforms of Ube1. Beta-actin was used as a loading control. (B) Top, Mobility shift detection of phosphorylated forms of Ube1 isoform E1A, 3 phosphate (3P), 2 phosphate (2P), and 1 phosphate (1P) in proliferating and quiescent B lymphocytes. Cell lysates were run on a 6% SDS-PAGE containing 50μM of acrylamide-coupled phosphate chelator (PhosTag) and analyzed by Western blotting with an antibody specific for the E1A isoform. Bottom, His-tagged Ube1 was purified from Hela cells, treated or not treated with λ phosphatase, and analyzed as described previously. The phosphatase-treated sample reveals the position of nonphosphorylated Ube1 (0P). Right, Quantification of the phosphorylated forms of Ube1 isoform E1A in proliferating and quiescent B lymphocytes and of purified Ube1 with ImageGauge software. Data are representative of 3 experiments and shown as mean ± SEM. (C) Effect of the Ube1-specific inhibitor Pyr-41 on repair of (6-4)PPs at 3 hours after irradiation in naive and differentiated THP1 leukemia cells, and in quiescent B lymphocytes (n = 6 donors).