OTUD1 interacts with NICD and cleaves ubiquitin from the K1770 site of NICD. (A) Confocal microscopy was used to analyze colocalization between OTUD1 and Notch2. (B) Jurkat E6.1 cells were transfected with pCDH-OTUD1 plasmids. Notch2 proteins were immunoprecipitated with an anti-Notch2 antibody and then OTUD1 and Notch2 protein levels were analyzed by western blot. (C) Jurkat E6.1 cells were stimulated with anti-CD3 (0.1 μg/mL) antibody for 24 or 48 hours in vitro. Notch2 proteins were immunoprecipitated with anti-Notch2 antibody and then OTUD1 and Notch2 were assessed by western blot. (D) Schematic diagrams of Notch2 full-length and Notch2 different mutants. (E) HEK293T cells were transfected with Myc-Notch2-FL, Myc-Notch2-ECD, Myc-Notch2-TMD and Myc- NICD. The colocalization between Notch2 and OTUD1 were evaluated by confocal. (F) HEK293T cells were transfected with Flag-NICD. OTUD1 proteins were immunoprecipitated with an anti-OTUD1 antibody and then OTUD1 and Flag-NICD were analyzed by western blot. (G) HEK293T cells were transfected with Myc-NICD, shCON or shOTUD1. Myc-NICD proteins were immunoprecipitated with anti-Myc beads and the ubiquitination levels of NICD were evaluated by western blot. (H) Schematic diagram of the ubiquitination K site of NICD protein. (I) HEK293T cells were transfected with Flag-NICD-WT, K1701R, K1703R, K1705R, K1738R, K1768R, K1770R, K2121R, together with pCDH-OTUD1 and HA-ub. Flag-NICD proteins were analyzed by immunoprecipitation and immunoblotting. Data in panels B-C,F-G,I are representative of 3 independent experiments and data in panels C,G,I are summarized as mean ± SD of 3 experiments. FL, full length.