Cdh1 depletion stabilizes USP1 during the G1 phase of the cell cycle. (A) T98G cells were transfected for 48 h with a control (Ctrl) siRNA (AllStars Negative; QIAGEN) or Cdh1 siRNA. Cells were serum starved in culture media containing 0.05% FBS for 24 h to arrest them in G0/G1. 0 indicates cells grown in regular media. (B) U2OS cells were transfected with the indicated siRNAs, synchronized in M phase (M) by incubating with nocodazole for 16 h, washed, and released in fresh media for 3 h for G1 phase (G1). Separate samples were collected for FACS. (C) U2OS cells were transfected with the indicated siRNAs and treated for the indicated time points with cycloheximide (CHX) to inhibit protein synthesis. (D) U2OS cells were transfected with the indicated siRNAs and synchronized in G1 as in B. Samples were collected and lysed according to protocols described for the UbVS DUB activity assay (see Materials and methods). Higher shift in the USP1 protein band indicates active USP1 (covalently modified USP1 by HA-UbVS). (E) Expression constructs were cotransfected in U2OS cells and treated for 6 h with 10 µM of the proteasome inhibitor MG132 before termination. Samples were lysed and collected for immunoprecipitation (IP) or loaded for input. (F) U2OS cells were transfected with Myc-USP1 wild type (WT) for 48 h and treated with MG132 as in E. Samples were lysed and collected for immunoprecipitation. *, heavy chain band. (G) T98G cells were serum starved as in A for the indicated times. Samples were lysed and collected for immunoprecipitation and input (10%) and probed with the indicated antibodies for Western blot analysis.