Figure 2.

Association of mammalian COP1 with ubiquitin and ubiquitinated protein in vivo. (A) Mammalian COP1 associates to ubiquitinated proteins in vivo. Lysates from 293 cells transfected with FLAG-tagged MmCOP1 and/or HA-tagged ubiquitin were subjected to immunoprecipitation by an anti-FLAG antibody, followed by immunoblotting by anti-HA and anti-FLAG antibodies as indicated. Molecular weights (in kDa) and IgG band are indicated on the left. IP, immunoprecipitation; IB, immunoblot. (B) Mammalian COP1 is a ubiquitination substrate. FLAG-tagged MmCOP1 or N280 and His-tagged ubiquitin were either co-transfected or individually transfected into 293 cells as indicated. His-tagged ubiquitinated protein was purified from the cell lysates under denaturing condition (in 8 M urea) by using Ni-NTA agarose beads. Immunoblot was performed by using an anti-FLAG antibody as indicated.