Fig. 2. NR inhibits the deubiquitinating activity of USP4 in HCT116 cells. (A) HCT116 cells were transfected with SRT-USP4 and HA-ubiquitin (HA-Ub). Transfected cells were or were not treated with 40 µM NR for 24 h, and then 10 µM MG132 was added for 4 h to block protein degradation. Ubiquitinated proteins were precipitated by anti-SRT antibody and detected using anti-K48 (left) and anti-K63 (right) ubiquitin antibodies. (B) HCT116 cells were treated with the indicated concentrations of NR for 24 h, and endogenous β-catenin expression was observed by immunoblotting (top). The immunoblot band of β-catenin was quantified by densitometry (bottom). The protein level of β-catenin was normalized to β-actin. (C) HCT116 cells were treated with 100 µg/ml cycloheximide (CHX) with or without 40 µM NR (top). Empty vector (control) and SRT-USP4 plasmid was transiently transfected into HCT116 cell. At 24h after transfection, the cells were treated with 100 µg/ml CHX with or without 40 µM NR for indicated times (bottom). The cells were lysed and the amount of β-catenin was analyzed by immunoblotting (left) and the expression amount was quantified by densitometry (right). (D) HCT116 cells were or were not treated with 10 µM or 20 µM NR for 24 h. The cells were then treated with 10 µM MG132 for 4 h prior to harvest. Endogenous ubiquitinated β-catenin was precipitated using anti-β-catenin antibody and then immunoblotted with ubiquitin antibody.